HUMAN ANTIBODIES THAT BIND HUMAN TNFalpha

ABSTRACT

Human antibodies, preferably recombinant human antibodies, that specifically bind to human tumor necrosis factor α (hTNFα) are disclosed. These antibodies have high affinity for hTNFα (e.g., K d =10 −8  M or less), a slow off rate for hTNFα dissociation (e.g., K off =10 −3  sec −1  or less) and neutralize hTNFα activity in vitro and in vivo. An antibody of the invention can be a full-length antibody or an antigen-binding portion thereof. The antibodies, or antibody portions, of the invention are useful for detecting hTNFα and for inhibiting hTNFα activity, e.g., in a human subject suffering from a disorder in which hTNFα activity is detrimental. Nucleic acids, vectors and host cells for expressing the recombinant human antibodies of the invention, and methods of synthesizing the recombinant human antibodies, are also encompassed by the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/369,451, filed onFeb. 11, 2009, which is a continuation of U.S. Ser. No. 11/787,901,filed on Apr. 17, 2007, now issued as U.S. Pat. No. 7,541,031, which isa continuation application of U.S. Ser. No. 09/801,185, filed on Mar. 7,2001, now issued as U.S. Pat. No. 7,223,394, which is a continuation ofU.S. Ser. No. 09/125,098 filed on Mar. 16, 1999, now issued as U.S. Pat.No. 6,258,562, which claims priority to International Application SerialNo. PCT/US97/02219 filed Feb. 10, 1997, which claims priority to U.S.provisional Application Ser. No. 60/031,476 filed Nov. 25, 1996.International Application Serial No. PCT/US97/02219 is also acontinuation-in-part of U.S. application Ser. No. 08/599,226 filed Feb.9, 1996. The contents of each of the above applications and patents areexpressly incorporated by reference herein.

BACKGROUND OF THE INVENTION

Tumor necrosis factor α (TNFα) is a cytokine produced by numerous celltypes, including monocytes and macrophages, that was originallyidentified based on its capacity to induce the necrosis of certain mousetumors (see e.g., Old, L. (1985) Science 230:630-632). Subsequently, afactor termed cachectin, associated with cachexia, was shown to be thesame molecule as TNFα. TNFα has been implicated in mediating shock (seee.g., Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57:505-518;Beutler, B. and Cerami, A. (1989) Annu. Rev. Immunol. 7:625-655).Furthermore, TNFα has been implicated in the pathophysiology of avariety of other human diseases and disorders, including sepsis,infections, autoimmune diseases, transplant rejection andgraft-versus-host disease (see e.g., Moeller, A., et al. (1990) Cytokine2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.; European PatentPublication No. 260 610 B1 by Moeller, A., et al. Vasilli, P. (1992)Annu. Rev. Immunol. 10:411-452; Tracey, K. J. and Cerami, A. (1994)Annu. Rev. Med. 45:491-503).

Because of the harmful role of human TNFα (hTNFα) in a variety of humandisorders, therapeutic strategies have been designed to inhibit orcounteract hTNFα activity. In particular, antibodies that bind to, andneutralize, hTNFα have been sought as a means to inhibit hTNFα activity.Some of the earliest of such antibodies were mouse monoclonal antibodies(mAbs), secreted by hybridomas prepared from lymphocytes of miceimmunized with hTNFα (see e.g., Hahn T; et al., (1985) Proc Natl AcadSci USA 82: 3814-3818; Liang, C-M., et al. (1986) Biochem. Biophys. Res.Commun. 137:847-854; Hirai, M., et al. (1987) J. Immunol. Methods96:57-62; Fendly, B. M., et al. (1987) Hybridoma 6:359-370; Moeller, A.,et al. (1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller etal.; European Patent Publication No. 186 833 B1 by Wallach, D.; EuropeanPatent Application Publication No. 218 868 A1 by Old et al.; EuropeanPatent Publication No. 260 610 B1 by Moeller, A., et al.). While thesemouse anti-hTNFα antibodies often displayed high affinity for hTNFα(e.g., Kd≦10⁻⁹M) and were able to neutralize hTNFα activity, their usein vivo may be limited by problems associated with administration ofmouse antibodies to humans, such as short serum half life, an inabilityto trigger certain human effector functions and elicitation of anunwanted immune response against the mouse antibody in a human (the“human anti-mouse antibody” (HAMA) reaction).

In an attempt to overcome the problems associated with use offully-murine antibodies in humans, murine anti-hTNFα antibodies havebeen genetically engineered to be more “human-like.” For example,chimeric antibodies, in which the variable regions of the antibodychains are murine-derived and the constant regions of the antibodychains are human-derived, have been prepared (Knight, D. M, et al.(1993) Mol. Immunol. 30:1443-1453; PCT Publication No. WO 92/16553 byDaddona, P. E., et al.). Additionally, humanized antibodies, in whichthe hypervariable domains of the antibody variable regions aremurine-derived but the remainder of the variable regions and theantibody constant regions are human-derived, have also been prepared(PCT Publication No. WO 92/11383 by Adair, J. R., et al.). However,because these chimeric and humanized antibodies still retain some murinesequences, they still may elicit an unwanted immune reaction, the humananti-chimeric antibody (HACA) reaction, especially when administered forprolonged periods, e.g., for chronic indications, such as rheumatoidarthritis (see e.g., Elliott, M. J., et al. (1994) Lancet 344:1125-1127;Elliot, M. J., et al. (1994) Lancet 344:1105-1110).

A preferred hTNFα inhibitory agent to murine mAbs or derivatives thereof(e.g., chimeric or humanized antibodies) would be an entirely humananti-hTNFα antibody, since such an agent should not elicit the HAMAreaction, even if used for prolonged periods. Human monoclonalautoantibodies against hTNFα have been prepared using human hybridomatechniques (Boyle, P., et al. (1993) Cell. Immunol. 152:556-568; Boyle,P., et al. (1993) Cell. Immunol. 152:569-581; European PatentApplication Publication No. 614 984 A2 by Boyle, et al.). However, thesehybridoma-derived monoclonal autoantibodies were reported to have anaffinity for hTNFα that was too low to calculate by conventionalmethods, were unable to bind soluble hTNFα and were unable to neutralizehTNFα-induced cytotoxicity (see Boyle, et al.; supra). Moreover, thesuccess of the human hybridoma technique depends upon the naturalpresence in human peripheral blood of lymphocytes producingautoantibodies specific for hTNFα. Certain studies have detected serumautoantibodies against hTNFα in human subjects (Fomsgaard, A., et al.(1989) Scand. J. Immunol. 30:219-223; Bendtzen, K., et al. (1990) Prog.Leukocyte Biol. 10B:447-452), whereas others have not (Leusch, H-G., etal. (1991) J. Immunol. Methods 139:145-147).

Alternative to naturally-occurring human anti-hTNFα antibodies would bea recombinant hTNFα antibody. Recombinant human antibodies that bindhTNFα with relatively low affinity (i.e., K_(d)˜10⁻⁷M) and a fast offrate (i.e., K_(off)˜10⁻² sec⁻¹) have been described (Griffiths, A. D.,et al. (1993) EMBO J. 12:725-734). However, because of their relativelyfast dissociation kinetics, these antibodies may not be suitable fortherapeutic use. Additionally, a recombinant human anti-hTNFα has beendescribed that does not neutralize hTNFα activity, but rather enhancesbinding of hTNFα to the surface of cells and enhances internalization ofhTNFα (Lidbury, A., et al. (1994) Biotechnol. Ther. 5:27-45; PCTPublication No. WO 92/03145 by Aston, R. et al.)

Accordingly, human antibodies, such as recombinant human antibodies,that bind soluble hTNFα with high affinity and slow dissociationkinetics and that have the capacity to neutralize hTNFα activity,including hTNFα-induced cytotoxicity (in vitro and in vivo) andhTNFα-induced cell activation, are still needed.

SUMMARY OF THE INVENTION

This invention provides human antibodies, preferably recombinant humanantibodies, that specifically bind to human TNFα. The antibodies of theinvention are characterized by binding to hTNFα with high affinity andslow dissociation kinetics and by neutralizing hTNFα activity, includinghTNFα-induced cytotoxicity (in vitro and in vivo) and hTNFα-inducedcellular activation. Antibodies of the invention are furthercharacterized by binding to hTNFα but not hTNFβ (lymphotoxin) and byhaving the ability to bind to other primate TNFαs and non-primate TNFαsin addition to human TNFα.

The antibodies of the invention can be full-length (e.g., an IgG1 orIgG4 antibody) or can comprise only an antigen-binding portion (e.g., aFab, F(ab′)₂ or scFv fragment). The most preferred recombinant antibodyof the invention, termed D2E7, has a light chain CDR3 domain comprisingthe amino acid sequence of SEQ ID NO: 3 and a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 4. Preferably, the D2E7antibody has a light chain variable region (LCVR) comprising the aminoacid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 2.

In one embodiment, the invention provides an isolated human antibody, oran antigen-binding portion thereof, that dissociates from human TNFαwith a K_(d) of 1×10⁻⁸ M or less and a K_(off) rate constant of 1×10⁻³s⁻¹ or less, both determined by surface plasmon resonance, andneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻⁷ M or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, dissociates from humanTNFα with a K_(off) of 5×10⁻⁴ s⁻¹ or less, or even more preferably, witha K_(off) of 1×10⁻⁴ s⁻¹ or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ Mor less, even more preferably with an IC₅₀ of 1×10⁻⁹ M or less and stillmore preferably with an IC₅₀ of 5×10⁻¹⁰ M or less.

In another embodiment, the invention provides a human antibody, orantigen-binding portion thereof, with the following characteristics:

a) dissociates from human TNFα with a K_(off) of 1×10⁻³ s⁻¹ or less, asdetermined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12.

More preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 5×10⁻⁴ s⁻¹ or less. Stillmore preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 1×10⁻⁴ s⁻¹ or less.

In yet another embodiment, the invention provides a human antibody, oran antigen-binding portion thereof, with an LCVR having CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8,and with an HCVR having a CDR3 domain comprising the amino acid sequenceof SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. More preferably,the LCVR further has a CDR2 domain comprising the amino acid sequence ofSEQ ID NO: 5 and the HCVR further has a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 6. Still more preferably, the LCVR furtherhas CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 andthe HCVR has a CDR1 domain comprising the amino acid sequence of SEQ IDNO: 8.

In still another embodiment, the invention provides an isolated humanantibody, or an antigen binding portion thereof, with an LCVR comprisingthe amino acid sequence of SEQ ID NO: 1 and an HCVR comprising the aminoacid sequence of SEQ ID NO: 2. In certain embodiments, the antibody hasan IgG1 heavy chain constant region or an IgG4 heavy chain constantregion. In yet other embodiments, the antibody is a Fab fragment, anF(ab′)₂ fragment or a single chain Fv fragment.

In still other embodiments, the invention provides antibodies, orantigen-binding portions thereof, with an LCVR having CDR3 domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or with an HCVR having a CDR3domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34 and SEQ ID NO: 35.

In yet another embodiment, the invention provides an isolated humanantibody, or antigen-binding portion thereof, that neutralizes theactivity of human TNFα but not human TNFβ (lymphotoxin). In a preferredembodiment, the human antibody, or antigen-binding portion thereof,neutralizes the activity of human TNFα, chimpanzee TNFα and at least oneadditional primate TNFα selected from the group consisting of baboonTNFα, marmoset TNFα, cynomolgus TNFα and rhesus TNFα. Preferably, theantibody also neutralizes the activity of at least one non-primate TNFα.For example, in one subembodiment, the isolated human antibody, orantigen-binding portion thereof, also neutralizes the activity of canineTNFα. In another subembodiment, the isolated human antibody, orantigen-binding portion thereof, also neutralizes the activity of pigTNFα. In yet another subembodiment, the isolated human antibody, orantigen-binding portion thereof, also neutralizes the activity of mouseTNFα.

Another aspect of the invention pertains to nucleic acid moleculesencoding the antibodies, or antigen-binding portions, of the invention.A preferred nucleic acid of the invention, encoding a D2E7 LCVR, has thenucleotide sequence shown in FIG. 7 and SEQ ID NO 36. Another preferrednucleic acid of the invention, encoding a D2E7 HCVR, has the nucleotidesequence shown in FIG. 8 and SEQ ID NO 37. Recombinant expressionvectors carrying the antibody-encoding nucleic acids of the invention,and host cells into which such vectors have been introduced, are alsoencompassed by the invention, as are methods of making the antibodies ofthe invention by culturing the host cells of the invention.

Yet another aspect of the invention pertains to methods for inhibitinghuman TNFα activity using an antibody, or antigen-binding portionthereof, of the invention. In one embodiment, the method comprisescontacting human TNFα with the antibody of the invention, orantigen-binding portion thereof, such that human TNFα activity isinhibited. In another embodiment, the method comprises administering anantibody of the invention, or antigen-binding portion thereof, to ahuman subject suffering from a disorder in which TNFα activity isdetrimental such that human TNFα activity in the human subject isinhibited. The disorder can be, for example, sepsis, an autoimmunedisease (e.g., rheumatoid arthritis, allergy, multiple sclerosis,autoimmune diabetes, autoimmune uveitis and nephrotic syndrome), aninfectious disease, a malignancy, transplant rejection orgraft-versus-host disease, a pulmonary disorder, a bone disorder, anintestinal disorder or a cardiac disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the amino acid sequences of the light chainvariable region of D2E7 (D2E7 VL; also shown in SEQ ID NO: 1),alanine-scan mutants of D2E7 VL (LD2E7*.A1, LD2E7*.A3, LD2E7*.A4,LD2E7*.A5, LD2E7*.A7 and LD2E7*.A8), the light chain variable region ofthe D2E7-related antibody 2SD4 (2SD4 VL; also shown in SEQ ID NO: 9) andother D2E7-related light chain variable regions (EP B12, VL10E4,VL100A9, VL100D2, VL10F4, LOE5, VLL0F9, VLL0F10, VLLOG7, VLLOG9, VLLOH1,VLLOH10, VL1B7, VL1C1, VL1C7, VL0.1F4, VL0.1H8, LOE7, LOE7.A andLOE7.T). FIG. 1A shows the FR1, CDR1, FR2 and CDR2 domains. FIG. 1Bshows the FR3, CDR3 and FR4 domains. The light chain CDR1 (“CDR L1”),CDR2 (“CDR L2”) and CDR3 (“CDR L3”) domains are boxed.

FIGS. 2A and 2B show the amino acid sequences of the heavy chainvariable region of D2E7 (D2E7 VH; also shown in SEQ ID NO: 2),alanine-scan mutants of D2E7 VH (HD2E7*.A1, HD2E7*.A2, HD2E7*.A3,HD2E7*.A4, HD2E7*.A5, HD2E7*.A6, HD2E7*.A7, HD2E7*.A8 and HD2E7*.A9),the heavy chain variable region of the D2E7-related antibody 2SD4 (2SD4VH; also shown in SEQ ID NO: 10) and other D2E7-related heavy chainvariable regions (VH1B11, VH1D8, VH1A11, VH1B12, VH1-D2, VH1E4, VH1F6,VH1G1, 3C—H2, VH1-D2.N and VH1-D2.Y).

FIG. 2A shows the FR1, CDR1, FR2 and CDR2 domains. FIG. 2B shows theFR3, CDR3 and FR4 domains. The heavy chain CDR1 (“CDR H1”), CDR2 (“CDRH2”) and CDR3 (“CDR H3”) domains are boxed.

FIG. 3 is a graph depicting the inhibition of TNFα-induced L929cytotoxicity by the human anti-hTNFα antibody D2E7, as compared to themurine anti-hTNFα antibody MAK 195.

FIG. 4 is a graph depicting the inhibition of rhTNFα binding to hTNFαreceptors on U-937 cells by the human anti-hTNFα antibody D2E7, ascompared to the murine anti-hTNFα antibody MAK 195.

FIG. 5 is a graph depicting the inhibition of TNFα-induced ELAM-1expression on HUVEC by the human anti-hTNFα antibody D2E7, as comparedto the murine anti-hTNFα antibody MAK 195.

FIG. 6 is a bar graph depicting protection from TNFα-induced lethalityin D-galactosamine-sensitized mice by administration of the humananti-hTNFα antibody D2E7 (black bars), as compared to the murineanti-hTNFα antibody MAK 195 (hatched bars).

FIG. 7 shows the nucleotide sequence of the light chain variable regionof D2E7, with the predicted amino acid sequence below the nucleotidesequence. The CDR L1, CDR L2 and CDR L3 regions are underlined.

FIG. 8 shows the nucleotide sequence of the heavy chain variable regionof D2E7, with the predicted amino acid sequence below the nucleotidesequence. The CDR H1, CDR H2 and CDR H3 regions are underlined.

FIG. 9 is a graph depicting the effect of D2E7 antibody treatment on themean joint size of Tg197 transgenic mice as a polyarthritis model.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to isolated human antibodies, or antigen-bindingportions thereof, that bind to human TNFα with high affinity, a low offrate and high neutralizing capacity. Various aspects of the inventionrelate to antibodies and antibody fragments, and pharmaceuticalcompositions thereof, as well as nucleic acids, recombinant expressionvectors and host cells for making such antibodies and fragments. Methodsof using the antibodies of the invention to detect human TNFα or toinhibit human TNFα activity, either in vitro or in vivo, are alsoencompassed by the invention.

In order that the present invention may be more readily understood,certain terms are first defined.

The term “human TNFα” (abbreviated herein as hTNFα, or simply hTNF), asused herein, is intended to refer to a human cytokine that exists as a17 kD secreted form and a 26 kD membrane associated form, thebiologically active form of which is composed of a trimer ofnoncovalently bound 17 kD molecules. The structure of hTNFα is describedfurther in, for example, Pennica, D., et al. (1984) Nature 312:724-729;Davis, J. M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E. Y.,et al. (1989) Nature 338:225-228. The term human TNFα is intended toinclude recombinant human TNFα (rhTNFα), which can be prepared bystandard recombinant expression methods or purchased commercially (R & DSystems, Catalog No. 210-TA, Minneapolis, Minn.).

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region is comprised of one domain, CL. The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., hTNFα). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesin molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesinmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesinmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther in Section II, below), antibodies isolated from a recombinant,combinatorial human antibody library (described further in Section III,below), antibodies isolated from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes (see e.g., Taylor, L. D., etal. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared,expressed, created or isolated by any other means that involves splicingof human immunoglobulin gene sequences to other DNA sequences. Suchrecombinant human antibodies have variable and constant regions derivedfrom human germline immunoglobulin sequences. In certain embodiments,however, such recombinant human antibodies are subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe VH and VL regions of the recombinant antibodies are sequences that,while derived from and related to human germline VH and VL sequences,may not naturally exist within the human antibody germline repertoire invivo.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hTNFα is substantially free of antibodies that specifically bindantigens other than hTNFα). An isolated antibody that specifically bindshTNFα may, however, have cross-reactivity to other antigens, such asTNFα molecules from other species (discussed in further detail below).Moreover, an isolated antibody may be substantially free of othercellular material and/or chemicals.

A “neutralizing antibody”, as used herein (or an “antibody thatneutralized hTNFα activity”), is intended to refer to an antibody whosebinding to hTNFα results in inhibition of the biological activity ofhTNFα. This inhibition of the biological activity of hTNFα can beassessed by measuring one or more indicators of hTNFα biologicalactivity, such as hTNFα-induced cytotoxicity (either in vitro or invivo), hTNFα-induced cellular activation and hTNFα binding to hTNFαreceptors. These indicators of hTNFα biological activity can be assessedby one or more of several standard in vitro or in vivo assays known inthe art (see Example 4). Preferably, the ability of an antibody toneutralize hTNFα activity is assessed by inhibition of hTNFα-inducedcytotoxicity of L929 cells. As an additional or alternative parameter ofhTNFα activity, the ability of an antibody to inhibit hTNFα-inducedexpression of ELAM-1 on HUVEC, as a measure of hTNFα-induced cellularactivation, can be assessed.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Example 1 and Jönsson, U., et al. (1993) Ann.Biol. Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; andJohnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule”, as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., VH, VL,CDR3) that bind hTNFα, is intended to refer to a nucleic acid moleculein which the nucleotide sequences encoding the antibody or antibodyportion are free of other nucleotide sequences encoding antibodies orantibody portions that bind antigens other than hTNFα, which othersequences may naturally flank the nucleic acid in human genomic DNA.Thus, for example, an isolated nucleic acid of the invention encoding aVH region of an anti-TNFα antibody contains no other sequences encodingother VH regions that bind antigens other than TNFα.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Human Antibodies that Bind Human TNFα

This invention provides isolated human antibodies, or antigen-bindingportions thereof, that bind to human TNFα with high affinity, a low offrate and high neutralizing capacity. Preferably, the human antibodies ofthe invention are recombinant, neutralizing human anti-hTNFα antibodies.The most preferred recombinant, neutralizing antibody of the inventionis referred to herein as D2E7 and has VL and VH sequences as shown inFIGS. 1A, 1B and FIGS. 2A, 2B, respectively (the amino acid sequence ofthe D2E7 VL region is also shown in SEQ ID NO: 1; the amino acidsequence of the D2E7 VH region is also shown in SEQ ID NO: 2). Thebinding properties of D2E7, as compared to the murine anti-hTNFα MAK 195mAb that exhibits high affinity and slow dissociation kinetics andanother human anti-hTNFα antibody related in sequence to D2E7, 2SD4, aresummarized below:

K_(off) k_(on) Antibody sec⁻¹ M⁻¹ sec⁻¹ K_(d) M Stoichiometry D2E7 IgG18.81 × 10⁻⁵ 1.91 × 10⁵ 6.09 × 10⁻¹⁰ 1.2 2SD4 IgG4  8.4 × 10⁻³ 4.20 × 10⁵2.00 × 10⁻⁸  0.8 MAK 195 8.70 × 10⁻⁵ 1.90 × 10⁵ 4.60 × 10⁻¹⁰ 1.4 F(ab′)₂

The D2E7 antibody, and related antibodies, also exhibit a strongcapacity to neutralize hTNFα activity, as assessed by several in vitroand in vivo assays (see Example 4). For example, these antibodiesneutralize hTNFα-induced cytotoxicity of L929 cells with IC₅₀ values inthe range of about 10⁻⁷ M to about 10⁻¹⁰ M. D2E7, when expressed as afull-length IgG1 antibody, neutralizes hTNFα-induced cytotoxicity ofL929 cells with IC₅₀ of about 1.25×10⁻¹⁰ M. Moreover, the neutralizingcapacity of D2E7 is maintained when the antibody is expressed as a Fab,F(ab′)₂ or scFv fragment. D2E7 also inhibits TNFα-induced cellularactivation, as measured by hTNFα-induced ELAM-1 expression on HUVEC(IC₅₀=about 1.85×10⁻¹⁰ M), and binding of hTNFα to hTNFα receptors onU-937 cells (IC₅₀=about 1.56×10⁻¹⁰ M). Regarding the latter, D2E7inhibits the binding of hTNFα to both the p55 and p75 hTNFα receptors.Furthermore, the antibody inhibits hTNFα-induced lethality in vivo inmice (ED₅₀=1-2.5 μg/mouse).

Regarding the binding specificity of D2E7, this antibody binds to humanTNFα in various forms, including soluble hTNFα, transmembrane hTNFα andhTNFα bound to cellular receptors. D2E7 does not specifically bind toother cytokines, such as lymphotoxin (TNFβ), IL-1α, IL-1β, IL-2, L-4,L-6, L-8, IFNγ and TGFβ. However, D2E7 does exhibit crossreactivity totumor necrosis factors from other species. For example, the antibodyneutralizes the activity of at least five primate TNFαs (chimpanzee,baboon, marmoset, cynomolgus and rhesus) with approximately equivalentIC₅₀ values as for neutralization of hTNFα (see Example 4, subsectionE). D2E7 also neutralizes the activity of mouse TNFα, althoughapproximately 1000-fold less well than human TNFα (see Example 4,subsection E). D2E7 also binds to canine and porcine TNFα.

In one aspect, the invention pertains to D2E7 antibodies and antibodyportions, D2E7-related antibodies and antibody portions, and other humanantibodies and antibody portions with equivalent properties to D2E7,such as high affinity binding to hTNFα with low dissociation kineticsand high neutralizing capacity. In one embodiment, the inventionprovides an isolated human antibody, or an antigen-binding portionthereof, that dissociates from human TNFα with a K_(d) of 1×10⁻⁸ M orless and a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determinedby surface plasmon resonance, and neutralizes human TNFα cytotoxicity ina standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁷ M or less. Morepreferably, the isolated human antibody, or antigen-binding portionthereof, dissociates from human TNFα with a K_(off) of 5×10⁻⁴ s⁻¹ orless, or even more preferably, with a K_(off) of 1×10⁻⁴ s⁻¹ or less.More preferably, the isolated human antibody, or antigen-binding portionthereof, neutralizes human TNFα cytotoxicity in a standard in vitro L929assay with an IC₅₀ of 1×10⁻⁸ M or less, even more preferably with anIC₅₀ of 1×10⁻⁹ M or less and still more preferably with an IC₅₀ of5×10⁻¹⁰ M or less. In a preferred embodiment, the antibody is anisolated human recombinant antibody, or an antigen-binding portionthereof. In another preferred embodiment, the antibody also neutralizesTNFα-induced cellular activation, as assessed using a standard in vitroassay for TNFα-induced ELAM-1 expression on human umbilical veinendothelial cells (HUVEC).

Surface plasmon resonance analysis for determining K_(d) and K_(off) canbe performed as described in Example 1. A standard in vitro L929 assayfor determining IC₅₀ values is described in Example 4, subsection A. Astandard in vitro assay for TNFα-induced ELAM-1 expression on humanumbilical vein endothelial cells (HUVEC) is described in Example 4,subsection C. Examples of recombinant human antibodies that meet, or arepredicted to meet, the aforementioned kinetic and neutralizationcriteria include antibodies having the following [VH/VL] pairs, thesequences of which are shown in FIGS. 1A, 1B, 2A and 2B (see alsoExamples 2, 3 and 4 for kinetic and neutralization analyses): [D2E7VH/D2E7 VL]; [HD2E7*.A1/D2E7 VL], [HD2E7*.A2/D2E7 VL], [HD2E7*.A3/D2E7VL], [HD2E7*.A4/D2E7 VL], [HD2E7*.A5/D2E7 VL], [HD2E7*.A6/D2E7 VL],[HD2E7*.A7/D2E7 VL], [HD2E7*.A8/D2E7 VL], [HD2E7*.A9/D2E7 VL], [D2E7VH/LD2E7*.A1], [D2E7 VH/LD2E7*.A4], [D2E7 VH/LD2E7*.A5], [D2E7VH/LD2E7*.A7], [D2E7 VH/LD2E7*.A8], [HD2E7*.A9/LD2E7*.A1],[VH1-D2/LOE7], [VH1-D2.N/LOE7.T], [VH1-D2.Y/LOE7.A], [VH1-D2.N/LOE7.A],[VH1-D2/EP B12] and [3C—H2/LOE7].

It is well known in the art that antibody heavy and light chain CDR3domains play an important role in the binding specificity/affinity of anantibody for an antigen. Accordingly, in another aspect, the inventionpertains to human antibodies that have slow dissociation kinetics forassociation with hTNFα and that have light and heavy chain CDR3 domainsthat structurally are identical to or related to those of D2E7. Asdemonstrated in Example 3, position 9 of the D2E7 VL CDR3 can beoccupied by Ala or Thr without substantially affecting the K_(off).Accordingly, a consensus motif for the D2E7 VL CDR3 comprises the aminoacid sequence: Q-R—Y—N—R-A-P—Y-(T/A) (SEQ ID NO: 3). Additionally,position 12 of the D2E7 VH CDR3 can be occupied by Tyr or Asn, withoutsubstantially affecting the K_(off). Accordingly, a consensus motif forthe D2E7 VH CDR3 comprises the amino acid sequence:V—S—Y-L-S-T-A-S—S-L-D-(Y/N) (SEQ ID NO: 4). Moreover, as demonstrated inExample 2, the CDR3 domain of the D2E7 heavy and light chains isamenable to substitution with a single alanine residue (at position 1,4, 5, 7 or 8 within the VL CDR3 or at position 2, 3, 4, 5, 6, 8, 9, 10or 11 within the VH CDR3) without substantially affecting the K_(off).Still further, the skilled artisan will appreciate that, given theamenability of the D2E7 VL and VH CDR3 domains to substitutions byalanine, substitution of other amino acids within the CDR3 domains maybe possible while still retaining the low off rate constant of theantibody, in particular substitutions with conservative amino acids. A“conservative amino acid substitution”, as used herein, is one in whichone amino acid residue is replaced with another amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art, including basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Preferably, nomore than one to five conservative amino acid substitutions are madewithin the D2E7 VL and/or VH CDR3 domains. More preferably, no more thanone to three conservative amino acid substitutions are made within theD2E7 VL and/or VH CDR3 domains. Additionally, conservative amino acidsubstitutions should not be made at amino acid positions critical forbinding to hTNFα. As shown in Example 3, positions 2 and 5 of the D2E7VL CDR3 and positions 1 and 7 of the D2E7 VH CDR3 appear to be criticalfor interaction with hTNFα and thus, conservative amino acidsubstitutions preferably are not made at these positions (although analanine substitution at position 5 of the D2E7 VL CDR3 is acceptable, asdescribed above).

Accordingly, in another embodiment, the invention provides an isolatedhuman antibody, or antigen-binding portion thereof, with the followingcharacteristics:

a) dissociates from human TNFα with a K_(off) rate constant of 1×10⁻³s⁻¹ or less, as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12.

More preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 5×10⁻⁴ s⁻¹ or less. Evenmore preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 1×10⁻⁴ s⁻¹ or less.

In yet another embodiment, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, with a light chainvariable region (LCVR) having a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a singlealanine substitution at position 1, 4, 5, 7 or 8, and with a heavy chainvariable region (HCVR) having a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11.Preferably, the LCVR further has a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 5 (i.e., the D2E7 VL CDR2) and the HCVR furtherhas a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6(i.e., the D2E7 VH CDR2). Even more preferably, the LCVR further hasCDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 (i.e.,the D2E7 VL CDR1) and the HCVR has a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 8 (i.e., the D2E7 VH CDR1). The frameworkregions for VL preferably are from the V_(κ)I human germline family,more preferably from the A20 human germline Vk gene and most preferablyfrom the D2E7 VL framework sequences shown in FIGS. 1A and 1B. Theframework regions for VH preferably are from the V_(H)3 human germlinefamily, more preferably from the DP-31 human germline VH gene and mostpreferably from the D2E7 VH framework sequences shown in FIGS. 2A and2B.

In still another embodiment, the invention provides an isolated humanantibody, or an antigen binding portion thereof, with a light chainvariable region (LCVR) comprising the amino acid sequence of SEQ ID NO:1 (i.e., the D2E7 VL) and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 2 (i.e., the D2E7 VH).In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constantregion. Preferably, the heavy chain constant region is an IgG1 heavychain constant region or an IgG4 heavy chain constant region.Furthermore, the antibody can comprise a light chain constant region,either a kappa light chain constant region or a lambda light chainconstant region. Preferably, the antibody comprises a kappa light chainconstant region. Alternatively, the antibody portion can be, forexample, a Fab fragment or a single chain Fv fragment.

In still other embodiments, the invention provides an isolated humanantibody, or an antigen-binding portions thereof, having D2E7-related VLand VH CDR3 domains, for example, antibodies, or antigen-bindingportions thereof, with a light chain variable region (LCVR) having aCDR3 domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 or with a heavychain variable region (HCVR) having a CDR3 domain comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 4, SEQ IDNO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.

In yet another embodiment, the invention provides a recombinant humanantibody, or antigen-binding portion thereof, that neutralizes theactivity of human TNFα but not human TNFβ. Preferably, antibody, orantigen-binding portion thereof, also neutralizes the activity ofchimpanzee TNFα and at least one additional primate TNFα selected fromthe group consisting of baboon TNFα, marmoset TNFα, cynomolgus TNFα andrhesus TNFα. Preferably, the antibody, or antigen-binding portionthereof, neutralizes the human, chimpanzee and/or additional primateTNFα in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ M or less,more preferably 1×10⁻⁹ M or less, and even more preferably 5×10⁻¹⁰ M orless. In one subembodiment, the antibody also neutralizes the activityof canine TNFα, preferably in a standard in vitro L929 assay with anIC₅₀ of 1×10⁻⁷ M or less, more preferably 1×10⁻⁸ M or less and even morepreferably 5×10⁻⁹ M or less. In another subembodiment, the antibody alsoneutralizes the activity of pig TNFα, preferably with an IC₅₀ of 1×10⁻⁵M or less, more preferably 1×10⁻⁶ M or less and even more preferably5×10⁻⁷ M or less. In yet another embodiment, the antibody alsoneutralizes the activity of mouse TNFα, preferably with an IC₅₀ of1×10⁻⁴ M or less, more preferably 1×10⁻⁵ M or less and even morepreferably 5×10⁻⁶ M or less.

An antibody or antibody portion of the invention can be derivatized orlinked to another functional molecule (e.g., another peptide orprotein). Accordingly, the antibodies and antibody portions of theinvention are intended to include derivatized and otherwise modifiedforms of the human anti-hTNFα antibodies described herein, includingimmunoadhesin molecules. For example, an antibody or antibody portion ofthe invention can be functionally linked (by chemical coupling, geneticfusion, noncovalent association or otherwise) to one or more othermolecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detectable agent, a cytotoxic agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociate of the antibody or antibody portion with another molecule(such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which an antibody or antibody portion ofthe invention may be derivatized include fluorescent compounds.Exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

II. Expression of Antibodies

An antibody, or antibody portion, of the invention can be prepared byrecombinant expression of immunoglobulin light and heavy chain genes ina host cell. To express an antibody recombinantly, a host cell istransfected with one or more recombinant expression vectors carrying DNAfragments encoding the immunoglobulin light and heavy chains of theantibody such that the light and heavy chains are expressed in the hostcell and, preferably, secreted into the medium in which the host cellsare cultured, from which medium the antibodies can be recovered.Standard recombinant DNA methodologies are used obtain antibody heavyand light chain genes, incorporate these genes into recombinantexpression vectors and introduce the vectors into host cells, such asthose described in Sambrook, Fritsch and Maniatis (eds), MolecularCloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,(1989), Ausubel, F. M. et al. (eds.) Current Protocols in MolecularBiology, Greene Publishing Associates, (1989) and in U.S. Pat. No.4,816,397 by Boss et al.

To express D2E7 or a D2E7-related antibody, DNA fragments encoding thelight and heavy chain variable regions are first obtained. These DNAscan be obtained by amplification and modification of germline light andheavy chain variable sequences using the polymerase chain reaction(PCR). Germline DNA sequences for human heavy and light chain variableregion genes are known in the art (see e.g., the “Vbase” human germlinesequence database; see also Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson, I.M., et al. (1992) “The Repertoire of Human Germline V_(H) SequencesReveals about Fifty Groups of V_(H) Segments with DifferentHypervariable Loops” J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al.(1994) “A Directory of Human Germ-line V_(κ) Segments Reveals a StrongBias in their Usage” Eur. J. Immunol. 24:827-836; the contents of eachof which are expressly incorporated herein by reference). To obtain aDNA fragment encoding the heavy chain variable region of D2E7, or aD2E7-related antibody, a member of the V_(H)3 family of human germlineVH genes is amplified by standard PCR. Most preferably, the DP-31 VHgermline sequence is amplified. To obtain a DNA fragment encoding thelight chain variable region of D2E7, or a D2E7-related antibody, amember of the V_(κ)I family of human germline VL genes is amplified bystandard PCR. Most preferably, the A20 VL germline sequence isamplified. PCR primers suitable for use in amplifying the DP-31 germlineVH and A20 germline VL sequences can be designed based on the nucleotidesequences disclosed in the references cited supra, using standardmethods.

Once the germline VH and VL fragments are obtained, these sequences canbe mutated to encode the D2E7 or D2E7-related amino acid sequencesdisclosed herein. The amino acid sequences encoded by the germline VHand VL DNA sequences are first compared to the D2E7 or D2E7-related VHand VL amino acid sequences to identify amino acid residues in the D2E7or D2E7-related sequence that differ from germline. Then, theappropriate nucleotides of the germline DNA sequences are mutated suchthat the mutated germline sequence encodes the D2E7 or D2E7-relatedamino acid sequence, using the genetic code to determine whichnucleotide changes should be made. Mutagenesis of the germline sequencesis carried out by standard methods, such as PCR-mediated mutagenesis (inwhich the mutated nucleotides are incorporated into the PCR primers suchthat the PCR product contains the mutations) or site-directedmutagenesis.

Moreover, it should be noted that if the “germline” sequences obtainedby PCR amplification encode amino acid differences in the frameworkregions from the true germline configuration (i.e., differences in theamplified sequence as compared to the true germline sequence, forexample as a result of somatic mutation), it may be desirable to changethese amino acid differences back to the true germline sequences (i.e.,“backmutation” of framework residues to the germline configuration).

Once DNA fragments encoding D2E7 or D2E7-related VH and VL segments areobtained (by amplification and mutagenesis of germline VH and VL genes,as described above), these DNA fragments can be further manipulated bystandard recombinant DNA techniques, for example to convert the variableregion genes to full-length antibody chain genes, to Fab fragment genesor to a scFv gene. In these manipulations, a VL- or VH-encoding DNAfragment is operatively linked to another DNA fragment encoding anotherprotein, such as an antibody constant region or a flexible linker. Theterm “operatively linked”, as used in this context, is intended to meanthat the two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the VH-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but most preferably is a kappaconstant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

To express the antibodies, or antibody portions of the invention, DNAsencoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or, more typically, both genesare inserted into the same expression vector. The antibody genes areinserted into the expression vector by standard methods (e.g., ligationof complementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present).Prior to insertion of the D2E7 or D2E7-related light or heavy chainsequences, the expression vector may already carry antibody constantregion sequences. For example, one approach to converting the D2E7 orD2E7-related VH and VL sequences to full-length antibody genes is toinsert them into expression vectors already encoding heavy chainconstant and light chain constant regions, respectively, such that theVH segment is operatively linked to the CH segment(s) within the vectorand the VL segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in-frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to includes promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, seee.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NS0 myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain (but notboth) of an antibody of this invention. Recombinant DNA technology mayalso be used to remove some or all of the DNA encoding either or both ofthe light and heavy chains that is not necessary for binding to hTNFα.The molecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies of the invention. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the invention and the other heavy and lightchain are specific for an antigen other than hTNFα by crosslinking anantibody of the invention to a second antibody by standard chemicalcrosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to enhancer/promoter regulatory elements (e.g., derived fromSV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLPpromoter regulatory element or an SV40 enhancer/AdMLP promoterregulatory element) to drive high levels of transcription of the genes.The recombinant expression vector also carries a DHFR gene, which allowsfor selection of CHO cells that have been transfected with the vectorusing methotrexate selection/amplification. The selected transformanthost cells are culture to allow for expression of the antibody heavy andlight chains and intact antibody is recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium.

In view of the foregoing, another aspect of the invention pertains tonucleic acid, vector and host cell compositions that can be used forrecombinant expression of the antibodies and antibody portions of theinvention. The nucleotide sequence encoding the D2E7 light chainvariable region is shown in FIG. 7 and SEQ ID NO: 36. The CDR1 domain ofthe LCVR encompasses nucleotides 70-102, the CDR2 domain encompassesnucleotides 148-168 and the CDR3 domain encompasses nucleotides 265-291.The nucleotide sequence encoding the D2E7 heavy chain variable region isshown in FIG. 8 and SEQ ID NO: 37. The CDR1 domain of the HCVRencompasses nucleotides 91-105, the CDR2 domain encompasses nucleotides148-198 and the CDR3 domain encompasses nucleotides 295-330. It will beappreciated by the skilled artisan that nucleotide sequences encodingD2E7-related antibodies, or portions thereof (e.g., a CDR domain, suchas a CDR3 domain), can be derived from the nucleotide sequences encodingthe D2E7 LCVR and HCVR using the genetic code and standard molecularbiology techniques.

In one embodiment, the invention provides an isolated nucleic acidencoding a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3 (i.e., the D2E7 VL CDR3), or modified from SEQ ID NO: 3 bya single alanine substitution at position 1, 4, 5, 7 or 8 or by one tofive conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8and/or 9. This nucleic acid can encode only the CDR3 region or, morepreferably, encodes an entire antibody light chain variable region(LCVR). For example, the nucleic acid can encode an LCVR having a CDR2domain comprising the amino acid sequence of SEQ ID NO: 5 (i.e., theD2E7 VL CDR2) and a CDR1 domain comprising the amino acid sequence ofSEQ ID NO: 7 (i.e., the D2E7 VL CDR1).

In another embodiment, the invention provides an isolated nucleic acidencoding a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4 (i.e., the D2E7 VH CDR3), or modified from SEQ ID NO: 4 bya single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11or by one to five conservative amino acid substitutions at positions 2,3, 4, 5, 6, 8, 9, 10, 11 and/or 12. This nucleic acid can encode onlythe CDR3 region or, more preferably, encodes an entire antibody heavychain variable region (HCVR). For example, the nucleic acid can encode aHCVR having a CDR2 domain comprising the amino acid sequence of SEQ IDNO: 6 (i.e., the D2E7 VH CDR2) and a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 8 (i.e., the D2E7 VH CDR1).

In yet another embodiment, the invention provides isolated nucleic acidsencoding a D2E7-related CDR3 domain, e.g., comprising an amino acidsequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ IDNO: 34 and SEQ ID NO: 35.

In still another embodiment, the invention provides an isolated nucleicacid encoding an antibody light chain variable region comprising theamino acid sequence of SEQ ID NO: 1 (i.e., the D2E7 LCVR). Preferablythis nucleic acid comprises the nucleotide sequence of SEQ ID NO: 36,although the skilled artisan will appreciate that due to the degeneracyof the genetic code, other nucleotide sequences can encode the aminoacid sequence of SEQ ID NO: 1. The nucleic acid can encode only the LCVRor can also encode an antibody light chain constant region, operativelylinked to the LCVR. In one embodiment, this nucleic acid is in arecombinant expression vector.

In still another embodiment, the invention provides an isolated nucleicacid encoding an antibody heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 2 (i.e., the D2E7 HCVR). Preferablythis nucleic acid comprises the nucleotide sequence of SEQ ID NO: 37,although the skilled artisan will appreciate that due to the degeneracyof the genetic code, other nucleotide sequences can encode the aminoacid sequence of SEQ ID NO: 2. The nucleic acid can encode only the HCVRor can also encode a heavy chain constant region, operatively linked tothe HCVR. For example, the nucleic acid can comprise an IgG1 or IgG4constant region. In one embodiment, this nucleic acid is in arecombinant expression vector.

The invention also provides recombinant expression vectors encoding bothan antibody heavy chain and an antibody light chain. For example, in oneembodiment, the invention provides a recombinant expression vectorencoding:

a) an antibody light chain having a variable region comprising the aminoacid sequence of SEQ ID NO: 1 (i.e., the D2E7 LCVR); and

b) an antibody heavy chain having a variable region comprising the aminoacid sequence of SEQ ID NO: 2 (i.e., the D2E7 HCVR).

The invention also provides host cells into which one or more of therecombinant expression vectors of the invention have been introduced.Preferably, the host cell is a mammalian host cell, more preferably thehost cell is a CHO cell, an NS0 cell or a COS cell.

Still further the invention provides a method of synthesizing arecombinant human antibody of the invention by culturing a host cell ofthe invention in a suitable culture medium until a recombinant humanantibody of the invention is synthesized. The method can furthercomprise isolating the recombinant human antibody from the culturemedium.

III. Selection of Recombinant Human Antibodies

Recombinant human antibodies of the invention in addition to the D2E7 orD2E7-related antibodies disclosed herein can be isolated by screening ofa recombinant combinatorial antibody library, preferably a scFv phagedisplay library, prepared using human VL and VH cDNAs prepared from mRNAderived from human lymphocytes. Methodologies for preparing andscreening such libraries are known in the art. In addition tocommercially available kits for generating phage display libraries(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.27-9400-01; and the Stratagene SurJZAP™ phage display kit, catalog no.240612), examples of methods and reagents particularly amenable for usein generating and screening antibody display libraries can be found in,for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTPublication No. WO 92/18619; Dower et al. PCT Publication No. WO91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al.PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard etal. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990)348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.

In a preferred embodiment, to isolate human antibodies with highaffinity and a low off rate constant for hTNFα, a murine anti-hTNFαantibody having high affinity and a low off rate constant for hTNFα(e.g., MAK 195, the hybridoma for which has deposit number ECACC 87050801) is first used to select human heavy and light chain sequenceshaving similar binding activity toward hTNFα, using the epitopeimprinting, or guided selection, methods described in Hoogenboom et al.,PCT Publication No. WO 93/06213. The antibody libraries used in thismethod are preferably scFv libraries prepared and screened as describedin McCafferty et al., PCT Publication No. WO 92/01047, McCafferty etal., Nature (1990) 348:552-554; and Griffiths et al., (1993) EMBO J12:725-734. The scFv antibody libraries preferably are screened usingrecombinant human TNFα as the antigen.

Once initial human VL and VH segments are selected, “mix and match”experiments, in which different pairs of the initially selected VL andVH segments are screened for hTNFα binding, are performed to selectpreferred VL/VH pair combinations. Additionally, to further improve theaffinity and/or lower the off rate constant for hTNFα binding, the VLand VH segments of the preferred VL/VH pair(s) can be randomly mutated,preferably within the CDR3 region of VH and/or VL, in a processanalogous to the in vivo somatic mutation process responsible foraffinity maturation of antibodies during a natural immune response. Thisin vitro affinity maturation can be accomplished by amplifying VH and VLregions using PCR primers complimentary to the VH CDR3 or VL CDR3,respectively, which primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode VH and VL segments into which random mutations havebeen introduced into the VH and/or VL CDR3 regions. These randomlymutated VH and VL segments can be rescreened for binding to hTNFα andsequences that exhibit high affinity and a low off rate for hTNFαbinding can be selected.

The amino acid sequences of selected antibody heavy and light chains canbe compared to germline heavy and light chain amino acid sequences. Incases where certain framework residues of the selected VL and/or VHchains differ from the germline configuration (e.g., as a result ofsomatic mutation of the immunoglobulin genes used to prepare the phagelibrary), it may be desirable to “backmutate” the altered frameworkresidues of the selected antibodies to the germline configuration (i.e.,change the framework amino acid sequences of the selected antibodies sothat they are the same as the germline framework amino acid sequences).Such “backmutation” (or “germlining”) of framework residues can beaccomplished by standard molecular biology methods for introducingspecific mutations (e.g., site-directed mutagenesis; PCR-mediatedmutagenesis, and the like).

Following screening and isolation of an anti-hTNFα antibody of theinvention from a recombinant immunoglobulin display library, nucleicacid encoding the selected antibody can be recovered from the displaypackage (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can be further manipulated to create other antibodyforms of the invention (e.g., linked to nucleic acid encoding additionalimmunoglobulin domains, such as additional constant regions). To expressa recombinant human antibody isolated by screening of a combinatoriallibrary, the DNA encoding the antibody is cloned into a recombinantexpression vector and introduced into a mammalian host cells, asdescribed in further detail in Section II above.

IV. Pharmaceutical Compositions and Pharmaceutical Administration

The antibodies and antibody-portions of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody or antibody portion of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the antibody is administered by intravenous infusion orinjection. In another preferred embodiment, the antibody is administeredby intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying that yields apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. The proper fluidityof a solution can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prolongedabsorption of injectable compositions can be brought about by includingin the composition an agent that delays absorption, for example,monostearate salts and gelatin.

The antibodies and antibody-portions of the present invention can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the preferred route/mode of administration isintravenous injection or infusion. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. In certain embodiments, the active compoundmay be prepared with a carrier that will protect the compound againstrapid release, such as a controlled release formulation, includingimplants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, an antibody or antibody portion of the inventionmay be orally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion ofthe invention is coformulated with and/or coadministered with one ormore additional therapeutic agents that are useful for treatingdisorders in which TNFα activity is detrimental. For example, ananti-hTNFα antibody or antibody portion of the invention may becoformulated and/or coadministered with one or more additionalantibodies that bind other targets (e.g., antibodies that bind othercytokines or that bind cell surface molecules), one or more cytokines,soluble TNFα receptor (see e.g., PCT Publication No. WO 94/06476) and/orone or more chemical agents that inhibit hTNFα production or activity(such as cyclohexane-ylidene derivatives as described in PCT PublicationNo. WO 93/19751). Furthermore, one or more antibodies of the inventionmay be used in combination with two or more of the foregoing therapeuticagents. Such combination therapies may advantageously utilize lowerdosages of the administered therapeutic agents, thus avoiding possibletoxicities or complications associated with the various monotherapies.

Nonlimiting examples of therapeutic agents for rheumatoid arthritis withwhich an antibody, or antibody portion, of the invention can be combinedinclude the following: non-steroidal anti-inflammatory drug(s) (NSAIDs);cytokine suppressive anti-inflammatory drug(s) (CSAIDs);CDP-571/BAY-10-3356 (humanized anti-TNFα antibody; Celltech/Bayer); cA2(chimeric anti-TNFα antibody; Centocor); 75 kdTNFR-IgG (75 kD TNFreceptor-IgG fusion protein; Immunex; see e.g., Arthritis & Rheumatism(1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche);IDEC-CE9.1/SB 210396 (non-depleting primatized anti-CD4 antibody;IDEC/SmithKline; see e.g., Arthritis & Rheumatism (1995) Vol. 38, S185);DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins; Seragen; seee.g., Arthritis & Rheumatism (1993) Vol. 36, 1223); Anti-Tac (humanizedanti-IL-2Rα; Protein Design Labs/Roche); IL-4 (anti-inflammatorycytokine; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10,anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4agonists (e.g., agonist antibodies); IL-1RA (IL-1 receptor antagonist;Synergen/Amgen); TNF-bp/s-TNFR (soluble TNF binding protein; see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284; Amer.J. Physiol.—Heart and Circulatory Physiology (1995) Vol. 268, pp.37-42); R973401 (phosphodiesterase Type IV inhibitor; see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); MK-966(COX-2 Inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S81); Iloprost (see e.g., Arthritis & Rheumatism (1996)Vol. 39, No. 9 (supplement), S82); methotrexate; thalidomide (see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S282) andthalidomide-related drugs (e.g., Celgen); leflunomide (anti-inflammatoryand cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39,No. 9 (supplement), S131; Inflammation Research (1996) Vol. 45, pp.103-107); tranexamic acid (inhibitor of plasminogen activation; seee.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284);T-614 (cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S282); prostaglandin E1 (see e.g., Arthritis &Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); Tenidap(non-steroidal anti-inflammatory drug; see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S280); Naproxen (non-steroidalanti-inflammatory drug; see e.g., Neuro Report (1996) Vol. 7, pp.1209-1213); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen(non-steroidal anti-inflammatory drug); Piroxicam (non-steroidalanti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatorydrug); Indomethacin (non-steroidal anti-inflammatory drug);Sulfasalazine (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S281); Azathioprine (see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S281); ICE inhibitor (inhibitor ofthe enzyme interleukin-1β converting enzyme); zap-70 and/or lckinhibitor (inhibitor of the tyrosine kinase zap-70 or lck); VEGFinhibitor and/or VEGF-R inhibitor (inhibitors of vascular endothelialcell growth factor or vascular endothelial cell growth factor receptor;inhibitors of angiogenesis); corticosteroid anti-inflammatory drugs(e.g., SB203580); TNF-convertase inhibitors; anti-IL-12 antibodies;interleukin-11 (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S296); interleukin-13 (see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S308); interleukin-17 inhibitors(see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),S120); gold; penicillamine; chloroquine; hydroxychloroquine;chlorambucil; cyclophosphamide; cyclosporine; total lymphoidirradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins;orally-administered peptides and collagen; lobenzarit disodium; CytokineRegulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals,Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10;T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycanpolysulphate; minocycline; anti-IL2R antibodies; marine and botanicallipids (fish and plant seed fatty acids; see e.g., DeLuca et al. (1995)Rheum. Dis. Clin. North Am. 21:759-777); auranofin; phenylbutazone;meclofenamic acid; flufenamic acid; intravenous immune globulin;zileuton; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus(rapamycin); amiprilose (therafectin); cladribine(2-chlorodeoxyadenosine); and azaribine.

Nonlimiting examples of therapeutic agents for inflammatory boweldisease with which an antibody, or antibody portion, of the inventioncan be combined include the following: budenoside; epidermal growthfactor; corticosteroids; cyclosporin, sulfasalazine; aminosalicylates;6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors;mesalamine; olsalazine; balsalazide; antioxidants; thromboxaneinhibitors; IL-1 receptor antagonists; anti-IL-1β monoclonal antibodies;anti-IL-6 monoclonal antibodies; growth factors; elastase inhibitors;pyridinyl-imidazole compounds; CDP-571/BAY-10-3356 (humanized anti-TNFαantibody; Celltech/Bayer); cA2 (chimeric anti-TNFα antibody; Centocor);75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see e.g.,Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol.44, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein;Hoffmann-LaRoche); interleukin-10 (SCH 52000; Schering Plough); IL-4;IL-10 and/or IL-4 agonists (e.g., agonist antibodies); interleukin-11;glucuronide- or dextran-conjugated prodrugs of prednisolone,dexamethasone or budesonide; ICAM-1 antisense phosphorothioateoligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); solublecomplement receptor 1 (TP10; T Cell Sciences, Inc.); slow-releasemesalazine; methotrexate; antagonists of Platelet Activating Factor(PAF); ciprofloxacin; and lignocaine.

Nonlimiting examples of therapeutic agents for multiple sclerosis withwhich an antibody, or antibody portion, of the invention can be combinedinclude the following: corticosteroids; prednisolone;methylprednisolone; azathioprine; cyclophosphamide; cyclosporine;methotrexate; 4-aminopyridine; tizanidine; interferon-β1a (Avonex™;Biogen); interferon-β1b (Betaseron™; Chiron/Berlex); Copolymer 1 (Cop-1;Copaxone™; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen;intravenous immunoglobulin; clabribine; CDP-571/BAY-10-3356 (humanizedanti-TNFα antibody; Celltech/Bayer); cA2 (chimeric anti-TNFα antibody;Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein;Immunex; see e.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J.Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNFreceptor-IgG fusion protein; Hoffmann-LaRoche); L-10; IL-4; and L-10and/or IL-4 agonists (e.g., agonist antibodies).

Nonlimiting examples of therapeutic agents for sepsis with which anantibody, or antibody portion, of the invention can be combined includethe following: hypertonic saline solutions; antibiotics; intravenousgamma globulin; continuous hemofiltration; carbapenems (e.g.,meropenem); antagonists of cytokines such as TNFα, IL-1β, IL-6 and/orIL-8; CDP-571/BAY-10-3356 (humanized anti-TNFα antibody;Celltech/Bayer); cA2 (chimeric anti-TNFα antibody; Centocor); 75kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see e.g.,Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol.44, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein;Hoffmann-LaRoche); Cytokine Regulating Agents (CRAs) HP228 and HP466(Houghten Pharmaceuticals, Inc.); SK&F 107647 (low molecular peptide;SmithKline Beecham); tetravalent guanylhydrazone CNI-1493 (PicowerInstitute); Tissue Factor Pathway Inhibitor (TFPI; Chiron); PHP(chemically modified hemoglobin; APEX Bioscience); iron chelators andchelates, including diethylenetriamine pentaacetic acid—iron (III)complex (DTPA iron (III); Molichem Medicines); lisofylline (syntheticsmall molecule methylxanthine; Cell Therapeutics, Inc.); PGG-Glucan(aqeuous soluble β1,3glucan; Alpha-Beta Technology); apolipoprotein A-1reconstituted with lipids; chiral hydroxamic acids (syntheticantibacterials that inhibit lipid A biosynthesis); anti-endotoxinantibodies; E5531 (synthetic lipid A antagonist; Eisai America, Inc.);rBPI₂₁ (recombinant N-terminal fragment of humanBactericidal/Permeability-Increasing Protein); and SyntheticAnti-Endotoxin Peptides (SAEP; BiosYnth Research Laboratories);

Nonlimiting examples of therapeutic agents for adult respiratorydistress syndrome (ARDS) with which an antibody, or antibody portion, ofthe invention can be combined include the following: anti-IL-8antibodies; surfactant replacement therapy; CDP-571/BAY-10-3356(humanized anti-TNFα antibody; Celltech/Bayer); cA2 (chimeric anti-TNFαantibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusionprotein; Immunex; see e.g., Arthritis & Rheumatism (1994) Vol. 37, S295;J. Invest. Med. (1996) Vol. 44, 235A); and 55 kdTNFR-IgG (55 kD TNFreceptor-IgG fusion protein; Hoffmann-LaRoche).

The use of the antibodies, or antibody portions, of the invention incombination with other therapeutic agents is discussed further insubsection IV.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody or antibody portion to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to benoted that dosage values may vary with the type and severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

IV. Uses of the Antibodies of the Invention

Given their ability to bind to hTNFα, the anti-hTNFα antibodies, orportions thereof, of the invention can be used to detect hTNFα (e.g., ina biological sample, such as serum or plasma), using a conventionalimmunoassay, such as an enzyme linked immunosorbent assays (ELISA), anradioimmunoassay (RIA) or tissue immunohistochemistry. The inventionprovides a method for detecting hTNFα in a biological sample comprisingcontacting a biological sample with an antibody, or antibody portion, ofthe invention and detecting either the antibody (or antibody portion)bound to hTNFα or unbound antibody (or antibody portion), to therebydetect hTNFα in the biological sample. The antibody is directly orindirectly labeled with a detectable substance to facilitate detectionof the bound or unbound antibody. Suitable detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ¹²⁵I,¹³¹I, ³⁵S or ³H.

Alternative to labeling the antibody, hTNFα can be assayed in biologicalfluids by a competition immunoassay utilizing rhTNFα standards labeledwith a detectable substance and an unlabeled anti-hTNFα antibody. Inthis assay, the biological sample, the labeled rhTNFα standards and theanti-hTNFα antibody are combined and the amount of labeled rhTNFαstandard bound to the unlabeled antibody is determined. The amount ofhTNFα in the biological sample is inversely proportional to the amountof labeled rhTNFα standard bound to the anti-hTNFα antibody.

A D2E7 antibody of the invention can also be used to detect TNFαs fromspecies other than humans, in particular TNFαs from primates (e.g.,chimpanzee, baboon, marmoset, cynomolgus and rhesus), pig and mouse,since D2E7 can bind to each of these TNFαs (discussed further in Example4, subsection E).

The antibodies and antibody portions of the invention are capable ofneutralizing hTNFα activity both in vitro and in vivo (see Example 4).Moreover, at least some of the antibodies of the invention, such asD2E7, can neutralize TNFα activity from other species. Accordingly, theantibodies and antibody portions of the invention can be used to inhibitTNFα activity, e.g., in a cell culture containing hTNFα, in humansubjects or in other mammalian subjects having TNFαs with which anantibody of the invention cross-reacts (e.g. chimpanzee, baboon,marmoset, cynomolgus and rhesus, pig or mouse). In one embodiment, theinvention provides a method for inhibiting TNFα activity comprisingcontacting TNFα with an antibody or antibody portion of the inventionsuch that TNFα activity is inhibited. Preferably, the TNFα is humanTNFα. For example, in a cell culture containing, or suspected ofcontaining hTNFα, an antibody or antibody portion of the invention canbe added to the culture medium to inhibit hTNFα activity in the culture.

In another embodiment, the invention provides a method for inhibitingTNFα activity in a subject suffering from a disorder in which TNFαactivity is detrimental. TNFα has been implicated in the pathophysiologyof a wide variety of disorders (see e.g., Moeller, A., et al. (1990)Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.; EuropeanPatent Publication No. 260 610 B1 by Moeller, A.). The inventionprovides methods for TNFα activity in a subject suffering from such adisorder, which method comprises administering to the subject anantibody or antibody portion of the invention such that TNFα activity inthe subject is inhibited. Preferably, the TNFα is human TNFα and thesubject is a human subject. Alternatively, the subject can be a mammalexpressing a TNFα with which an antibody of the invention cross-reacts.Still further the subject can be a mammal into which has been introducedhTNFα (e.g., by administration of hTNFα or by expression of an hTNFαtransgene). An antibody of the invention can be administered to a humansubject for therapeutic purposes (discussed further below). Moreover, anantibody of the invention can be administered to a non-human mammalexpressing a TNFα with which the antibody cross-reacts (e.g., a primate,pig or mouse) for veterinary purposes or as an animal model of humandisease. Regarding the latter, such animal models may be useful forevaluating the therapeutic efficacy of antibodies of the invention(e.g., testing of dosages and time courses of administration).

As used herein, the term “a disorder in which TNFα activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of TNFα in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which TNFαactivity is detrimental is a disorder in which inhibition of TNFαactivity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of TNFα in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofTNFα in serum, plasma, synovial fluid, etc. of the subject), which canbe detected, for example, using an anti-TNFα antibody as describedabove. There are numerous examples of disorders in which TNFα activityis detrimental. The use of the antibodies and antibody portions of theinvention in the treatment of specific disorders is discussed furtherbelow:

A. Sepsis

Tumor necrosis factor has an established role in the pathophysiology ofsepsis, with biological effects that include hypotension, myocardialsuppression, vascular leakage syndrome, organ necrosis, stimulation ofthe release of toxic secondary mediators and activation of the clottingcascade (see e.g., Moeller, A., et al. (1990) Cytokine 2:162-169; U.S.Pat. No. 5,231,024 to Moeller et al.; European Patent Publication No.260 610 B1 by Moeller, A.; Tracey, K. J. and Cerami, A. (1994) Annu.Rev. Med. 45:491-503; Russell, D. and Thompson, R. C. (1993) Curr. Opin.Biotech. 4:714-721). Accordingly, the human antibodies, and antibodyportions, of the invention can be used to treat sepsis in any of itsclinical settings, including septic shock, endotoxic shock, gramnegative sepsis and toxic shock syndrome.

Furthermore, to treat sepsis, an anti-hTNFα antibody, or antibodyportion, of the invention can be coadministered with one or moreadditional therapeutic agents that may further alleviate sepsis, such asan interleukin-1 inhibitor (such as those described in PCT PublicationNos. WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (see e.g.,PCT Publication No. WO 93/11793) or an antagonist of platelet activatingfactor (see e.g., European Patent Application Publication No. EP 374510). Other combination therapies for the treatment of sepsis arediscussed further in subsection III.

Additionally, in a preferred embodiment, an anti-TNFα antibody orantibody portion of the invention is administered to a human subjectwithin a subgroup of sepsis patients having a serum or plasmaconcentration of IL-6 above 500 pg/ml, and more preferably 1000 pg/ml,at the time of treatment (see PCT Publication No. WO 95/20978 by Daum,L., et al.).

B. Autoimmune Diseases

Tumor necrosis factor has been implicated in playing a role in thepathophysiology of a variety of autoimmune diseases. For example, TNFαhas been implicated in activating tissue inflammation and causing jointdestruction in rheumatoid arthritis (see e.g., Moeller, A., et al.(1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.;European Patent Publication No. 260 610 B1 by Moeller, A.; Tracey andCerami, supra; Arend, W. P. and Dayer, J-M. (1995) Arth. Rheum.38:151-160; Fava, R. A., et al. (1993) Clin. Exp. Immunol. 94:261-266).TNFα also has been implicated in promoting the death of islet cells andin mediating insulin resistance in diabetes (see e.g., Tracey andCerami, supra; PCT Publication No. WO 94/08609). TNFα also has beenimplicated in mediating cytotoxicity to oligodendrocytes and inductionof inflammatory plaques in multiple sclerosis (see e.g., Tracey andCerami, supra). Chimeric and humanized murine anti-hTNFα antibodies haveundergone clinical testing for treatment of rheumatoid arthritis (seee.g., Elliott, M. J., et al. (1994) Lancet 344:1125-1127; Elliot, M. J.,et al. (1994) Lancet 344:1105-1110; Rankin, E. C., et al. (1995) Br. J.Rheumatol. 34:334-342).

The human antibodies, and antibody portions of the invention can be usedto treat autoimmune diseases, in particular those associated withinflammation, including rheumatoid arthritis, rheumatoid spondylitis,osteoarthritis and gouty arthritis, allergy, multiple sclerosis,autoimmune diabetes, autoimmune uveitis and nephrotic syndrome.Typically, the antibody, or antibody portion, is administeredsystemically, although for certain disorders, local administration ofthe antibody or antibody portion at a site of inflammation may bebeneficial (e.g., local administration in the joints in rheumatoidarthritis or topical application to diabetic ulcers, alone or incombination with a cyclohexane-ylidene derivative as described in PCTPublication No. WO 93/19751). An antibody, or antibody portion, of theinvention also can be administered with one or more additionaltherapeutic agents useful in the treatment of autoimmune diseases, asdiscussed further in subsection III.

C. Infectious Diseases

Tumor necrosis factor has been implicated in mediating biologicaleffects observed in a variety of infectious diseases. For example, TNFαhas been implicated in mediating brain inflammation and capillarythrombosis and infarction in malaria. TNFα also has been implicated inmediating brain inflammation, inducing breakdown of the blood-brainbarrier, triggering septic shock syndrome and activating venousinfarction in meningitis. TNFα also has been implicated in inducingcachexia, stimulating viral proliferation and mediating central nervoussystem injury in acquired immune deficiency syndrome (AIDS).Accordingly, the antibodies, and antibody portions, of the invention,can be used in the treatment of infectious diseases, including bacterialmeningitis (see e.g., European Patent Application Publication No. EP 585705), cerebral malaria, AIDS and AIDS-related complex (ARC) (see e.g.,European Patent Application Publication No. EP 230 574), as well ascytomegalovirus infection secondary to transplantation (see e.g.,Fietze, E., et al. (1994) Transplantation 58:675-680). The antibodies,and antibody portions, of the invention, also can be used to alleviatesymptoms associated with infectious diseases, including fever andmyalgias due to infection (such as influenza) and cachexia secondary toinfection (e.g., secondary to AIDS or ARC).

D. Transplantation

Tumor necrosis factor has been implicated as a key mediator of allograftrejection and graft versus host disease (GVHD) and in mediating anadverse reaction that has been observed when the rat antibody OKT3,directed against the T cell receptor CD3 complex, is used to inhibitrejection of renal transplants (see e.g., Eason, J. D., et al. (1995)Transplantation 59:300-305; Suthanthiran, M. and Strom, T. B. (1994) NewEngl. J. Med. 331:365-375). Accordingly, the antibodies, and antibodyportions, of the invention, can be used to inhibit transplant rejection,including rejections of allografts and xenografts and to inhibit GVHD.Although the antibody or antibody portion may be used alone, morepreferably it is used in combination with one or more other agents thatinhibit the immune response against the allograft or inhibit GVHD. Forexample, in one embodiment, an antibody or antibody portion of theinvention is used in combination with OKT3 to inhibit OKT3-inducedreactions. In another embodiment, an antibody or antibody portion of theinvention is used in combination with one or more antibodies directed atother targets involved in regulating immune responses, such as the cellsurface molecules CD25 (interleukin-2 receptor-α), CD11a (LFA-1), CD54(ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2). In yetanother embodiment, an antibody or antibody portion of the invention isused in combination with one or more general immunosuppressive agents,such as cyclosporin A or FK506.

E. Malignancy

Tumor necrosis factor has been implicated in inducing cachexia,stimulating tumor growth, enhancing metastatic potential and mediatingcytotoxicity in malignancies. Accordingly, the antibodies, and antibodyportions, of the invention, can be used in the treatment ofmalignancies, to inhibit tumor growth or metastasis and/or to alleviatecachexia secondary to malignancy. The antibody, or antibody portion, maybe administered systemically or locally to the tumor site.

F. Pulmonary Disorders

Tumor necrosis factor has been implicated in the pathophysiology ofadult respiratory distress syndrome (ARDS), including stimulatingleukocyte-endothelial activation, directing cytotoxicity to pneumocytesand inducing vascular leakage syndrome. Accordingly, the antibodies, andantibody portions, of the invention, can be used to treat variouspulmonary disorders, including adult respiratory distress syndrome (seee.g., PCT Publication No. WO 91/04054), shock lung, chronic pulmonaryinflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis andsilicosis. The antibody, or antibody portion, may be administeredsystemically or locally to the lung surface, for example as an aerosol.An antibody, or antibody portion, of the invention also can beadministered with one or more additional therapeutic agents useful inthe treatment of pulmonary disorders, as discussed further in subsectionIII.

G. Intestinal Disorders

Tumor necrosis factor has been implicated in the pathophysiology ofinflammatory bowel disorders (see e.g., Tracy, K. J., et al. (1986)Science 234:470-474; Sun, X-M., et al. (1988) J. Clin. Invest.81:1328-1331; MacDonald, T. T., et al. (1990) Clin. Exp. Immunol.81:301-305). Chimeric murine anti-hTNFα antibodies have undergoneclinical testing for treatment of Crohn's disease (van Dullemen, H. M.,et al. (1995) Gastroenterology 109:129-135). The human antibodies, andantibody portions, of the invention, also can be used to treatintestinal disorders, such as idiopathic inflammatory bowel disease,which includes two syndromes, Crohn's disease and ulcerative colitis. Anantibody, or antibody portion, of the invention also can be administeredwith one or more additional therapeutic agents useful in the treatmentof intestinal disorders, as discussed further in subsection III.

H. Cardiac Disorders

The antibodies, and antibody portions, of the invention, also can beused to treat various cardiac disorders, including ischemia of the heart(see e.g., European Patent Application Publication No. EP 453 898) andheart insufficiency (weakness of the heart muscle)(see e.g., PCTPublication No. WO 94/20139).

I. Others

The antibodies, and antibody portions, of the invention, also can beused to treat various other disorders in which TNFα activity isdetrimental. Examples of other diseases and disorders in which TNFαactivity has been implicated in the pathophysiology, and thus which canbe treated using an antibody, or antibody portion, of the invention,include inflammatory bone disorders and bone resorption disease (seee.g., Bertolini, D. R., et al. (1986) Nature 319:516-518; Konig, A., etal. (1988) J. Bone Miner. Res. 3:621-627; Lerner, U. H. and Ohlin, A.(1993) J. Bone Miner. Res. 8:147-155; and Shankar, G. and Stern, P. H.(1993) Bone 14:871-876), hepatitis, including alcoholic hepatitis (seee.g., McClain, C. J. and Cohen, D. A. (1989) Hepatology 9:349-351;Felver, M. E., et al. (1990) Alcohol. Clin. Exp. Res. 14:255-259; andHansen, J., et al. (1994) Hepatology 20:461-474), viral hepatitis(Sheron, N., et al. (1991) J. Hepatol. 12:241-245; and Hussain, M. J.,et al. (1994) J. Clin. Pathol. 47:1112-1115), and fulminant hepatitis;coagulation disturbances (see e.g., van der Poll, T., et al. (1990) N.Engl. J. Med. 322:1622-1627; and van der Poll, T., et al. (1991) Prog.Clin. Biol. Res. 367:55-60), burns (see e.g., Giroir, B. P., et al.(1994) Am. J. Physiol. 267:H118-124; and Liu, X. S., et al. (1994) Burns20:40-44), reperfusion injury (see e.g., Scales, W. E., et al. (1994)Am. J. Physiol. 267:G1122-1127; Serrick, C., et al. (1994)Transplantation 58:1158-1162; and Yao, Y. M., et al. (1995)Resuscitation 29:157-168), keloid formation (see e.g., McCauley, R. L.,et al. (1992) J. Clin. Immunol. 12:300-308), scar tissue formation;pyrexia; periodontal disease; obesity and radiation toxicity.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

Example 1 Kinetic Analysis of Binding of Human Antibodies to hTNFα

Real-time binding interactions between ligand (biotinylated recombinanthuman TNFα (rhTNFα) immobilized on a biosensor matrix) and analyte(antibodies in solution) were measured by surface plasmon resonance(SPR) using the BIAcore system (Pharmacia Biosensor, Piscataway, N.J.).The system utilizes the optical properties of SPR to detect alterationsin protein concentrations within a dextran biosensor matrix. Proteinsare covalently bound to the dextran matrix at known concentrations.Antibodies are injected through the dextran matrix and specific bindingbetween injected antibodies and immobilized ligand results in anincreased matrix protein concentration and resultant change in the SPRsignal. These changes in SPR signal are recorded as resonance units (RU)and are displayed with respect to time along the y-axis of a sensorgram.

To facilitate immobilization of biotinylated rhTNFα on the biosensormatrix, streptavidin is covalently linked via free amine groups to thedextran matrix by first activating carboxyl groups on the matrix with100 mM N-hydroxysuccinimide (NHS) and 400 mMN-ethyl-N′-(3-diethylaminopropyl) carbodiimide hydrochloride (EDC).Next, streptavidin is injected across the activated matrix. Thirty-fivemicroliters of streptavidin (25 μg/ml), diluted in sodium acetate, pH4.5, is injected across the activated biosensor and free amines on theprotein are bound directly to the activated carboxyl groups. Unreactedmatrix EDC-esters are deactivated by an injection of 1 M ethanolamine.Streptavidin-coupled biosensor chips also are commercially available(Pharmacia BR-1000-16, Pharmacia Biosensor, Piscataway, N.J.).

Biotinylated rhTNFα was prepared by first dissolving 5.0 mg of biotin(D-biotinyl-ε-aminocaproic acid N-hydroxysuccinimide ester; BoehringerMannheim Cat. No. 1008 960) in 500 μl dimethylsulfoxide to make a 10mg/ml solution. Ten microliters of biotin was added per ml of rhTNFα (at2.65 mg/ml) for a 2:1 molar ratio of biotin to rhTNFα. The reaction wasmixed gently and incubated for two hours at room temperature in thedark. A PD-10 column, Sephadex G-25M (Pharmacia Catalog No. 17-0851-01)was equilibrated with 25 ml of cold PBS and loaded with 2 ml ofrhTNFα-biotin per column. The column was eluted with 10×1 ml cold PBS.Fractions were collected and read at OD280 (1.0 OD=1.25 mg/ml). Theappropriate fractions were pooled and stored at −80° C. until use.Biotinylated rhTNFα also is commercially available (R & D SystemsCatalog No. FTA00, Minneapolis, Minn.).

Biotinylated rhTNFα to be immobilized on the matrix via streptavidin wasdiluted in PBS running buffer (Gibco Cat. No. 14190-144, Gibco BRL,Grand Island, N.Y.) supplemented with 0.05% (BIAcore) surfactant P20(Pharmacia BR-1000-54, Pharmacia Biosensor, Piscataway, N.J.). Todetermine the capacity of rhTNFα-specific antibodies to bind immobilizedrhTNFα, a binding assay was conducted as follows. Aliquots ofbiotinylated rhTNFα (25 nM; 10 μl aliquots) were injected through thestreptavidin-coupled dextran matrix at a flow rate of 5 μl/min. Beforeinjection of the protein and immediately afterward, PBS buffer aloneflowed through each flow cell. The net difference in signal betweenbaseline and approximately 30 sec. after completion of biotinylatedrhTNFα injection was taken to represent the binding value (approximately500 RU). Direct rhTNFα-specific antibody binding to immobilizedbiotinylated rhTNFα was measured. Antibodies (20 μg/ml) were diluted inPBS running buffer and 25 μl aliquots were injected through theimmobilized protein matrices at a flow rate of 5 μl/min. Prior toinjection of antibody, and immediately afterwards, PBS buffer aloneflowed through each flow cell. The net difference in baseline signal andsignal after completion of antibody injection was taken to represent thebinding value of the particular sample. Biosensor matrices wereregenerated using 100 mM HCl before injection of the next sample. Todetermine the off rate (K_(off)), on rate (K_(on)), association rate(K_(a)) and dissociation rate (K_(d)) constants, BIAcore kineticevaluation software (version 2.1) was used.

Representative results of D2E7 (IgG4 full-length antibody) binding tobiotinylated rhTNFα, as compared to the mouse mAb MAK 195 (F(ab′)₂fragment), are shown below in Table 1.

TABLE 1 Binding of D2E7 IgG4 or MAK 195 to Biotinylated rhTNFα [Ab],rhTNFα, Ab, bound, rhTNFα/ K_(off), sec⁻¹, Antibody nM bound, RUs RUs Ab(Avg) D2E7 267 373 1215 1.14 8.45 × 10⁻⁵ 133 420 1569 1.30 5.42 × 10⁻⁵67 434 1633 1.31 4.75 × 10⁻⁵ 33 450 1532 1.19 4.46 × 10⁻⁵ 17 460 12960.98 3.47 × 10⁻⁵ 8 486 936 0.67 2.63 × 10⁻⁵ 4 489 536 0.38 2.17 × 10⁻⁵ 2470 244 0.18 3.68 × 10⁻⁵ (4.38 × 10⁻⁵) MAK 195 400 375 881 1.20 5.38 ×10⁻⁵ 200 400 1080 1.38 4.54 × 10⁻⁵ 100 419 1141 1.39 3.54 × 10⁻⁵ 50 4271106 1.32 3.67 × 10⁻⁵ 25 446 957 1.09 4.41 × 10⁻⁵ 13 464 708 0.78 3.66 ×10⁻⁵ 6 474 433 0.47 7.37 × 10⁻⁵ 3 451 231 0.26 6.95 × 10⁻⁵ (4.94 × 10⁻⁵⁾

In a second series of experiments, the molecular kinetic interactionsbetween an IgG1 full-length form of D2E7 and biotinylated rhTNF wasquantitatively analyzed using BIAcore technology, as described above,and kinetic rate constants were derived, summarized below in Tables 2, 3and 4.

TABLE 2 Apparent dissociation rate constants of the interaction betweenD2E7 and biotinylated rhTNF Experiment K_(d) (s⁻¹) 1 9.58 × 10⁻⁵ 2 9.26× 10⁻⁵ 3 7.60 × 10⁻⁵ Average 8.81 ± 1.06 × 10⁻⁵

TABLE 3 Apparent association rate constants of the interaction betweenD2E7 and biotinylated rhTNF Experiment K_(a) (M⁻¹, s⁻¹) 1 1.33 × 10⁵ 21.05 × 10⁵ 3 3.36 × 10⁵ Average 1.91 ± 1.26 × 10⁵

TABLE 4 Apparent kinetic reate and affinity constants of D2E7 andbiotinylated rhTNF Ex- periment K_(a) (M⁻¹, s⁻¹) K_(d) (s⁻¹) K_(d) (M) 11.33 × 10⁵ 9.58 × 10⁻⁵ 7.20 × 10⁻¹⁰ 2 1.05 × 10⁵ 9.26 × 10⁻⁵ 8.82 ×10⁻¹⁰ 3 3.36 × 10⁵ 7.60 × 10⁻⁵ 2.26 × 10⁻¹⁰ Average 1.91 ± 1.26 × 10⁵8.81 ± 1.06 × 10⁻⁵ 6.09 ± 3.42 × 10⁻¹⁰

Dissociation and association rate constants were calculated by analyzingthe dissociation and association regions of the sensorgrams by BIAanalysis software. Conventional chemical reaction kinetics were assumedfor the interaction between D2E7 and biotinylated rhTNF molecule: a zeroorder dissociation and first order association kinetics. For the sake ofanalysis, interaction only between one arm of the bivalent D2E7 antibodyand one unit of the trimeric biotinylated rhTNF was considered inchoosing molecular models for the analysis of the kinetic data. Threeindependent experiments were performed and the results were analyzedseparately. The average apparent dissociation rate constant (k_(d)) ofthe interaction between D2E7 and biotinylated rhTNF was 8.81±1.06×10⁻⁵s⁻¹, and the average apparent association rate constant, k_(a) was1.91±1.26×10⁵ M⁻¹ s⁻¹. The apparent intrinsic dissociation constant(K_(d)) was then calculated by the formula: K_(d)=k_(d)/k_(a). Thus, themean K_(d) of D2E7 antibody for rhTNF derived from kinetic parameterswas 6.09±3.42×10⁻¹⁰ M. Minor differences in the kinetic values for theIgG1 form of D2E7 (presented in Tables 2, 3 and 4) and the IgG4 form ofD2E7 (presented in Table 1 and in Examples 2 and 3) are not thought tobe true differences resulting from the presence of either an IgG1 or anIgG4 constant regions but rather are thought to be attributable to moreaccurate antibody concentration measurements used for the IgG1 kineticanalysis. Accordingly, the kinetic values for the IgG1 form of D2E7presented herein are thought to be the most accurate kinetic parametersfor the D2E7 antibody.

Example 2 Alanine Scanning Mutagenesis of D2E7 CDR3 Domains

A series of single alanine mutations were introduced by standard methodsalong the CDR3 domain of the D2E7 VL and the D2E7 VH regions. The lightchain mutations are illustrated in FIG. 1B (LD2E7*.A1, LD2E7*.A3,LD2E7*.A4, LD2E7*.A5, LD2E7*.A7 and LD2E7*.A8, having an alaninemutation at position 1, 3, 4, 5, 7 or 8, respectively, of the D2E7 VLCDR3 domain). The heavy chain mutations are illustrated in FIG. 2B(HD2E7*.A1, HD2E7*.A2, HD2E7*.A3, HD2E7*.A4, HD2E7*.A5, HD2E7*.A6,HD2E7*.A7, HD2E7*.A8 and HD2E7*.A9, having an alanine mutation atposition 2, 3, 4, 5, 6, 8, 9, 10 or 11, respectively, of the D2E7 VHCDR3 domain). The kinetics of rhTNFα interaction with an antibodycomposed of wild-type D2E7 VL and VH was compared to that of antibodiescomposed of 1) a wild-type D2E7 VL paired with an alanine-substitutedD2E7 VH; 2) a wild-type D2E7 VH paired with an alanine-substituted D2E7VL; or 3) an alanine-substituted D2E7 VL paired with analanine-substituted D2E7 VH. All antibodies were tested as full-length,IgG4 molecules.

Kinetics of interaction of antibodies with rhTNFα was determined bysurface plasmon resonance as described in Example 1. The K_(off) ratesfor the different VH/VL pairs are summarized below in Table 5:

TABLE 5 Binding of D2E7 Alanine-Scan Mutants to Biotinylated rhTNFα VHVL K_(off) (sec⁻¹) D2E7 VH D2E7 VL 9.65 × 10⁻⁵  HD2E7*.A1 D2E7 VL 1.4 ×10⁻⁴ HD2E7*.A2 D2E7 VL 4.6 × 10⁻⁴ HD2E7*.A3 D2E7 VL 8.15 × 10⁻⁴ HD2E7*.A4 D2E7 VL 1.8 × 10⁻⁴ HD2E7*.A5 D2E7 VL 2.35 × 10⁻⁴  HD2E7*.A6D2E7 VL 2.9 × 10⁻⁴ HD2E7*.A7 D2E7 VL 1.0 × 10⁻⁴ HD2E7*.A8 D2E7 VL 3.1 ×10⁻⁴ HD2E7*.A9 D2E7 VL 8.1 × 10⁻⁴ D2E7 VH LD2E7*.A1 6.6 × 10⁻⁵ D2E7 VHLD2E7*.A3 NOT DETECTABLE D2E7 VH LD2E7*.A4 1.75 × 10⁻⁴  D2E7 VHLD2E7*.A5 1.8 × 10⁻⁴ D2E7 VH LD2E7*.A7 1.4 × 10⁻⁴ D2E7 VH LD2E7*.A8 3.65× 10⁻⁴  HD2E7*.A9 LD2E7*.A1 1.05 × 10⁻⁴ 

These results demonstrate that the majority of positions of the CDR3domains of the D2E7 VL region and VH region are amenable to substitutionwith a single alanine residue. Substitution of a single alanine atposition 1, 4, 5, or 7 of the D2E7 VL CDR3 domain or at position 2, 5,6, 8, 9 or 10 of the D2E7 VH CDR3 domain does not significantly affectthe off rate of hTNFα binding as compared to the wild-type parental D2E7antibody. Substitution of alanine at position 8 of the D2E7 VL CDR3 orat position 3 of the D2E7 VH CDR3 gives a 4-fold faster K_(off) and analanine substitution at position 4 or 11 of D2E7 VH CDR3 gives an 8-foldfaster K_(off), indicating that these positions are more critical forbinding to hTNFα. However, a single alanine substitution at position 1,4, 5, 7 or 8 of the D2E7 VL CDR3 domain or at position 2, 3, 4, 5, 6, 8,9, 10 or 11 of the D2E7 VH CDR3 domain still results in an anti-hTNFαantibody having a K_(off) of 1×10⁻³ sec⁻¹ or less.

Example 3 Binding Analysis of D2E7-Related Antibodies

A series of antibodies related in sequence to D2E7 were analyzed fortheir binding to rhTNFα, as compared to D2E7, by surface plasmonresonance as described in Example 1. The amino acid sequences of the VLregions tested are shown in FIGS. 1A and 1B. The amino acid sequences ofthe VH regions tested are shown in FIGS. 2A and 2B. The K_(off) ratesfor various VH/VL pairs (in the indicated format, either as afull-length IgG1 or IgG4 antibody or as a scFv) are summarized below inTable 6:

TABLE 6 Binding of D2E7-Related Antibodies to Biotinylated rhTNFα VH VLFormat K_(off) (sec⁻¹) D2E7 VH D2E7 VL IgG1/IgG4 9.65 × 10⁻⁵ VH1-D2 LOE7IgG1/IgG4  7.7 × 10⁻⁵ VH1-D2 LOE7 scFv  4.6 × 10⁻⁴ VH1-D2.N LOE7.T IgG4 2.1 × 10⁻⁵ VH1-D2.Y LOE7.A IgG4  2.7 × 10⁻⁵ VH1-D2.N LOE7.A IgG4  3.2 ×10⁻⁵ VH1-D2 EP B12 scFv  8.0 × 10⁻⁴ VH1-D2 2SD4 VL scFv 1.94 × 10⁻³3C-H2 LOE7 scFv  1.5 × 10⁻³ 2SD4 VH LOE7 scFv 6.07 × 10⁻³ 2SD4 VH 2SD4VL scFv 1.37 × 10⁻² VH1A11 2SD4 VL scFv 1.34 × 10⁻² VH1B12 2SD4 VL scFv1.01 × 10⁻² VH1B11 2SD4 VL scFv  9.8 × 10⁻³ VH1E4 2SD4 VL scFv 1.59 ×10⁻² VH1F6 2SD4 VL scFv 2.29 × 10⁻² VH1D8 2SD4 VL scFv  9.5 × 10⁻³ VH1G12SD4 VL scFv 2.14 × 10⁻² 2SD4 VH EP B12 scFv  6.7 × 10⁻³ 2SD4 VH VL10E4scFv  9.6 × 10⁻³ 2SD4 VH VL100A9 scFv 1.33 × 10⁻² 2SD4 VH VL100D2 scFv1.41 × 10⁻² 2SD4 VH VL10F4 scFv 1.11 × 10⁻² 2SD4 VH VLLOE5 scFv 1.16 ×10⁻² 2SD4 VH VLL0F9 scFv 6.09 × 10⁻³ 2SD4 VH VLL0F10 scFv 1.34 × 10⁻²2SD4 VH VLLOG7 scFv 1.56 × 10⁻² 2SD4 VH VLLOG9 scFv 1.46 × 10⁻² 2SD4 VHVLLOH1 scFv 1.17 × 10⁻² 2SD4 VH VLLOH10 scFv 1.12 × 10⁻² 2SD4 VH VL1B7scFv  1.3 × 10⁻² 2SD4 VH VL1C1 scFv 1.36 × 10⁻² 2SD4 VH VL1C7 scFv  2.0× 10⁻² 2SD4 VH VL0.1F4 scFv 1.76 × 10⁻² 2SD4 VH VL0.1H8 scFv 1.14 × 10⁻²

The slow off rates (i.e., K_(off)≦1×10⁻⁴ sec⁻¹) for full-lengthantibodies (i.e., IgG format) having a VL selected from D2E7, LOE7,LOE7.T and LOE7.A, which have either a threonine or an alanine atposition 9, indicate that position 9 of the D2E7 VL CDR3 can be occupiedby either of these two residues without substantially affecting theK_(off). Accordingly, a consensus motif for the D2E7 VL CDR3 comprisesthe amino acid sequence: Q-R—Y—N—R-A-P—Y-(T/A) (SEQ ID NO: 3).Furthermore, the slow off rates (i.e., K_(off)≦1×10⁻⁴ sec⁻¹) forantibodies having a VH selected from D2E7, VH1-D2.N and VH1-D2.Y, whichhave either a tyrosine or an asparagine at position 12, indicate thatposition 12 of the D2E7 VH CDR3 can be occupied by either of these tworesidues without substantially affecting the K_(off). Accordingly, aconsensus motif for the D2E7 VH CDR3 comprises the amino acid sequence:V—S—Y-L-S-T-A-S—S-L-D-(Y/N) (SEQ ID NO: 4).

The results shown in Table 6 demonstrate that, in scFv format,antibodies containing the 2SD4 VL or VH CDR3 region exhibit a fasterK_(off) (i.e., K_(off)≧1×10⁻³ sec⁻¹) as compared to antibodiescontaining the D2E7 VL or VH CDR3 region. Within the VL CDR3, 2SD4differs from D2E7 at positions 2, 5 and 9. As discussed above, however,position 9 may be occupied by Ala (as in 2SD4) or Thr (as in D2E7)without substantially affecting the K_(off). Thus, by comparison of 2SD4and D2E7, positions 2 and 5 of the D2E7 VL CDR3, both arginines, can beidentified as being critical for the association of the antibody withhTNFα. These residues could be directly involved as contact residues inthe antibody binding site or could contribute critically to maintainingthe scaffolding architecture of the antibody molecule in this region.Regarding the importance of position 2, replacement of Arg (in LOE7,which has the same VL CDR3 as D2E7) with Lys (in EP B12) accelerates theoff rate by a factor of two. Regarding the importance of position 5,replacement of Arg (in D2E7) with Ala (in LD2E7*.A5), as described inExample 2, also accelerates the off rate two-fold. Furthermore, withouteither Arg at positions 2 and 5 (in 2SD4), the off rate is five-foldfaster. However, it should be noted that although position 5 isimportant for improved binding to hTNFα, a change at this position canbe negated by changes at other positions, as seen in VLLOE4, VLLOH1 orVL0.1H8.

Within the VH CDR3, 2SD4 differs from D2E7 at positions 1, 7 and 12. Asdiscussed above, however, position 12 may be occupied by Asn (as in2SD4) or Tyr (as in D2E7) without substantially affecting the K_(off).Thus, by comparison of 2SD4 and D2E7, positions 1 and 7 of the D2E7 VHCDR3 can be identified as being critical for binding to hTNFα. Asdiscussed above, these residues could be directly involved as contactresidues in the antibody binding site or could contribute critically tomaintaining the scaffolding architecture of the antibody molecule inthis region. Both positions are important for binding to hTNFα sincewhen the 3C—H2 VH CDR3 (which has a valine to alanine change at position1 with respect to the D2E7 VH CDR3) is used, the scFv has a 3-foldfaster off rate than when the D2E7 VH CDR3 is used but this off rate isstill four times slower than when the 2SD4 VH CDR3 is used (which haschanges at both positions 1 and 7 with respect to the D2E7 VH CDR3).

Example 4 Functional Activity of D2E7

To examine the functional activity of D2E7, the antibody was used inseveral assays that measure the ability of the antibody to inhibit hTNFαactivity, either in vitro or in vivo.

A. Neutralization of TNFα-Induced Cytotoxicity in L929 Cells

Human recombinant TNFα (rhTNFα) causes cell cytotoxicity to murine L929cells after an incubation period of 18-24 hours. Human anti-hTNFαantibodies were evaluated in L929 assays by coincubation of antibodieswith rhTNFα and the cells as follows. A 96-well microtiter platecontaining 100 μl of anti-hTNFα Abs was serially diluted ⅓ down theplate in duplicates using RPMI medium containing 10% fetal bovine serum(FBS). Fifty microliters of rhTNFα was added for a final concentrationof 500 pg/ml in each sample well. The plates were then incubated for 30minutes at room temperature. Next, 50 μl of TNFα-sensitive L929 mousefibroblasts cells were added for a final concentration of 5×10⁴ cellsper well, including 1 μg/ml Actinomycin-D. Controls included medium pluscells and rhTNFα plus cells. These controls, and a TNFα standard curve,ranging from 2 ng/ml to 8.2 pg/ml, were used to determine the quality ofthe assay and provide a window of neutralization. The plates were thenincubated overnight (18-24 hours) at 37° C. in 5% CO₂.

One hundred microliters of medium was removed from each well and 50 μlof 5 mg/ml 3,(4,4-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide(MTT; commercially available from Sigma Chemical Co., St. Louis, Mo.) inPBS was added. The plates were then incubated for 4 hours at 37° C.Fifty microliters of 20% sodium dodecyl sulfate (SDS) was then added toeach well and the plates were incubated overnight at 37° C. The opticaldensity at 570/630 nm was measured, curves were plotted for each sampleand IC₅₀s were determined by standard methods.

Representative results for human antibodies having various VL and VHpairs, as compared to the murine MAK 195 mAb, are shown in FIG. 3 and inTable 7 below.

TABLE 7 Neutralization of TNFα-Induced L929 Cytotoxicity VH VL StructureIC₅₀, M D2E7 D2E7 scFv 1.1 × 10⁻¹⁰ D2E7 D2E7 IgG4 4.7 × 10⁻¹¹ 2SD4 2SD4scFv/IgG1/IgG4 3.0 × 10⁻⁷ 2SD4 LOE7 scFv 4.3 × 10⁻⁸ VH1-D2 2SD4 scFv 1.0× 10⁻⁸ VH1-D2 LOE7 scFv/IgG1/IgG4 3.4 × 10⁻¹⁰ VH1.D2.Y LOE7.T IgG4 8.1 ×10⁻¹¹ VH1-D2.N LOE7.T IgG4 1.3 × 10⁻¹⁰ VH1-D2.Y LOE7.A IgG4 2.8 × 10⁻¹¹VH1-D2.N LOE7.A IgG4 6.2 × 10⁻¹¹ MAK 195 MAK 195 scFv 1.9 × 10⁻⁸ MAK 195MAK195 F(ab′)₂ 6.2 × 10⁻¹¹

The results in FIG. 3 and Table 7 demonstrate that the D2E7 humananti-hTNFα antibody, and various D2E7-related antibodies, neutralizeTNFα-induced L929 cytotoxicity with a capacity approximately equivalentto that of the murine anti-hTNFα mAb MAK 195.

In another series of experiments, the ability of the IgG1 form of D2E7to neutralize TNFα-induced L929 cytotoxicity was examined as describedabove. The results from three independent experiments, and the averagethereof, are summarized below in Table 8:

TABLE 8 Neutralization of TNFα-Induced L929 Cytotoxicity by D2E7 IgG1Experiment IC₅₀ [M] 1 1.26 × 10⁻¹⁰ 2 1.33 × 10⁻¹⁰ 3 1.15 × 10⁻¹⁰ Average1.25 ± 0.01 × 10⁻¹⁰

This series of experiments confirmed that D2E7, in the full-length IgG1form, neutralizes TNFα-induced L929 cytotoxicity with an average IC₅₀[M] of 1.25±0.01×10⁻¹⁰.

B. Inhibition of TNFα Binding to TNFα Receptors on U-937 Cells

The ability of human anti-hTNFα antibodies to inhibit the binding ofhTNFα to hTNFα receptors on the surface of cells was examined using theU-937 cell line (ATCC No. CRL 1593), a human histiocytic cell line thatexpresses hTNFα receptors. U-937 cells were grown in RPMI 1640 mediumsupplemented with 10% fetal bovine serum (Hyclone A-1111, HycloneLaboratories, Logan, Utah), L-glutamine (4 nM), HEPES buffer solution(10 mM), penicillin (100 μg/ml) and streptomycin (100 μg/ml). To examinethe activity of full-length IgG antibodies, U-937 cells werepreincubated with PBS supplemented with 1 mg/ml of human IgG (Sigma1-4506, Sigma Chemical Co., St. Louis, Mo.) for 45 minutes on ice andthen cells were washed three times with binding buffer. For the receptorbinding assay, U-937 cells (5×10⁶ cells/well) were incubated in abinding buffer (PBS supplemented with 0.2% bovine serum albumin) in96-well microtiter plates (Costar 3799, Costar Corp., Cambridge, Mass.)together with ¹²⁵I-labeled rhTNFα (3×10⁻¹⁰ M; 25 μCi/ml; obtained fromNEN Research Products, Wilmington, Del.), with or without anti-hTNFαantibodies, in a total volume of 0.2 ml. The plates were incubated onice for 1.5 hours. Then, 75 μl of each sample was transferred to 1.0 mltest tubes (Sarstedt 72.700, Sarstedt Corp., Princeton, N.J.) containingdibutylphthalate (Sigma D-2270, Sigma Chemical Co., St. Louis, Mo.) anddinonylphthalate (ICN 210733, ICN, Irvine, Calif.). The test tubescontained a 3001 mixture of dibutylphthalate and dinonylphthalate, 2:1volume ratio, respectively. Free (i.e., unbound) ¹²⁵I-labeled rhTNFα wasremoved by microcentrifugation for five minutes. Then, each test tubeend containing a cell pellet was cut with the aid of a microtube scissor(Bel-Art 210180001, Bel-Art Products, Pequannock, N.J.). The cell pelletcontains ¹²⁵I-labeled rhTNFα bound to the p60 or p80 TNFα receptor,whereas the aqueous phase above the oil mixture contains excess free¹²⁵I-labeled rhTNFα. All cell pellets were collected in a counting tube(Falcon 2052, Becton Dickinson Labware, Lincoln Park, N.J.) and countedin a scintillation counter.

Representative results are shown in FIG. 4. The IC₅₀ value for D2E7inhibition of hTNFα binding to hTNFα receptors on U-937 cells isapproximately 3×10⁻¹⁰ M in these experiments. These results demonstratethat the D2E7 human anti-hTNFα antibody inhibits rhTNFα binding to hTNFαreceptors on U-937 cells at concentrations approximately equivalent tothat of the murine anti-hTNFα mAb MAK 195.

In another series of experiments, the ability of the IgG1 form of D2E7to inhibit rhTNFα binding to hTNFα receptors on U-937 cells was examinedas described above. The results from three independent experiments, andthe average thereof, are summarized below in Table 9: 117813-04312

TABLE 9 Inhibition of TNF Receptor Binding on U-937 Cells by D2E7 IgG1Experiment IC₅₀ [M] 1 1.70 × 10⁻¹⁰ 2 1.49 × 10⁻¹⁰ 3 1.50 × 10⁻¹⁰ Average1.56 ± 0.12 × 10⁻¹⁰

This series of experiments confirmed that D2E7, in the full-length IgG1form, inhibits TNF receptor binding on U-937 cells with an average IC₅₀[M] of 1.56±0.12×10⁻¹⁰.

To investigate the inhibitory potency of D2E7 in the binding of¹²⁵I-rhTNF binding to individual p55 and p75 receptors, a solid phaseradioimmunoassay was performed. To measure the IC₅₀ values of D2E7 forseparate TNF receptors, varying concentrations of the antibody wereincubated with 3×10⁻¹⁰ concentration of ¹²⁵I-rhTNF. The mixture was thentested on separate plates containing either the p55 or the p75 TNFreceptors in a dose dependent manner. The results are summarized belowin Table 10:

TABLE 10 Inhibition of TNF Receptor Binding to p55 and p75 TNFR by D2E7IgG1 IC₅₀ [M] Reagent p55 TNFR p 75TNFR D2E7 1.47 × 10⁻⁹ 1.26 × 10⁻⁹rhTNF 2.31 × 10⁻⁹ 2.70 × 10⁻⁹

Inhibition of ¹²⁵I-rhTNF binding to the p55 and p75 TNF receptors onU937 cells by D2E7 followed a simple sigmoidal curve, indicating similarIC₅₀ values for each receptor. In the solid phase radioimmunoassay (RIA)experiments with recombinant TNF receptors, IC₅₀ values for inhibitionof ¹²⁵I-rhTNF binding to the p55 and the p75 receptors by D2E7 werecalculated as 1.47×10⁻⁹ and 1.26×10⁻⁹ M, respectively. The decrease inIC₅₀ values in the solid phase was probably due to higher density ofreceptors in the RIA format, as unlabeled rhTNF also inhibited withsimilar IC₅₀ values. The IC₅₀ values for inhibition of ¹²⁵I-rhTNFbinding to the p55 and the p75 receptors by unlabeled rhTNF were2.31×10⁻⁹ and 2.70×10⁻⁹ M, respectively

C. Inhibition of ELAM-1 Expression on HUVEC

Human umbilical vein endothelial cells (HUVEC) can be induced to expressendothelial cell leukocyte adhesion molecule 1 (ELAM-1) on theircell-surface by treatment with rhTNFα, which can be detected by reactingrhTNFα-treated HUVEC with an mouse anti-human ELAM-1 antibody. Theability of human anti-hTNFα antibodies to inhibit this TNFα-inducedexpression of ELAM-1 on HUVEC was examined as follows: HUVEC (ATCC No.CRL 1730) were plated in 96-well plates (5×10⁴ cells/well) and incubatedovernight at 37° C. The following day, serial dilutions of humananti-hTNFα antibody (1:10) were prepared in a microtiter plate, startingwith 20-100 μg/ml of antibody. A stock solution of rhTNFα was preparedat 4.5 ng/ml, aliquots of rhTNFα were added to each antibody-containingwell and the contents were mixed well. Controls included medium alone,medium plus anti-hTNFα antibody and medium plus rhTNFα. The HUVEC plateswere removed from their overnight incubation at 37° C. and the mediumgently aspirated from each well. Two hundred microliters of theantibody-rhTNFα mixture were transferred to each well of the HUVECplates. The HUVEC plates were then further incubated at 37° C. for 4hours. Next, a murine anti-ELAM-1 antibody stock was diluted 1:1000 inRPMI. The medium in each well of the HUVEC plate was gently aspirated,50 μl/well of the anti-ELAM-1 antibody solution was added and the HUVECplates were incubated 60 minutes at room temperature. An ¹²⁵I-labeledanti-mouse Ig antibody solution was prepared in RPMI (approximately50,000 cpm in 50 μl). The medium in each well of the HUVEC plates wasgently aspirated, the wells were washed twice with RPMI and 50 μl of the¹²⁵I-labeled anti-mouse Ig solution was added to each well. The plateswere incubated for one hour at room temperature and then each well waswashed three times with RPMI. One hundred eighty microliters of 5% SDSwas added to each well to lyse the cells. The cell lysate from each wellwas then transferred to a tube and counted in a scintillation counter.

Representative results are shown in FIG. 5. The IC₅₀ value for D2E7inhibition of hTNFα-induced expression of ELAM-1 on HUVEC isapproximately 6×10⁻¹¹ M in these experiments. These results demonstratethat the D2E7 human anti-hTNFα antibody inhibits the hTNFα-inducedexpression of ELAM-1 on HUVEC at concentrations approximately equivalentto that of the murine anti-hTNFα mAb MAK 195.

In another series of experiments, the ability of the IgG1 form of D2E7to inhibit hTNFα-induced expression of ELAM-1 on HUVEC was examined asdescribed above. The results from three independent experiments, and theaverage thereof, are summarized below in Table 11:

TABLE 11 Inhibition of TNFα-Induced ELAM-1 Expression by D2E7 IgG1Receptor Experiment IC₅₀ [M] 1 1.95 × 10⁻¹⁰ 2 1.69 × 10⁻¹⁰ 3 1.90 ×10⁻¹⁰ Average 1.85 ± 0.14 × 10⁻¹⁰

This series of experiments confirmed that D2E7, in the full-length IgG1form, inhibits TNFa-induced ELAM-1 expression on HUVEC with an averageIC₅₀ [M] of 1.85±0.14×10⁻¹⁰.

The neutralization potency of D2E7 IgG1 was also examined for the rhTNFinduced expression of two other adhesion molecules, ICAM-1 and VCAM-1.Since the rhTNF titration curve for ICAM-1 expression at 16 hours wasvery similar to the curve of ELAM-1 expression, the same concentrationof rhTNF was used in the antibody neutralization experiments. The HUVECwere incubated with rhTNF in the presence of varying concentrations ofD2E7 in a 37° C. CO₂ incubator for 16 hours, and the ICAM-1 expressionwas measured by mouse anti-ICAM-1 antibody followed by ¹²⁵I-labeledsheep anti-mouse antibody. Two independent experiments were performedand the IC₅₀ values were calculated. An unrelated human IgG1 antibodydid not inhibit the ICAM-1 expression.

The experimental procedure to test inhibition of VCAM-1 expression wasthe same as the procedure for ELAM-1 expression, except anti-VCAM-1 MAbwas used instead of anti-ELAM-1 MAb. Three independent experiments wereperformed and the IC₅₀ values were calculated. An unrelated human IgG1antibody did not inhibit VCAM-1 expression.

The results are summarized below in Table 12:

TABLE 12 Inhibition of ICAM-1 and VCAM-1 Expression byD2E7 IgG1 ICAM-1Inhibition IC₅₀ [M] Experiment IC₅₀ [M] Experiment IC₅₀ [M] 1 1.84 ×10⁻¹⁰ 1 1.03 × 10⁻¹⁰ 2 2.49 × 10⁻¹⁰ 2 9.26 × 10⁻¹¹ 3 1.06 × 10⁻¹⁰Average 2.17 ± 0.46 × 10⁻¹⁰ Average 1.01 ± 0.01 × 10⁻¹⁰

These experiments demonstrate that treatment of primary human umbilicalvein endothelial cells with rhTNF led to optimum expression of adhesionmolecules: ELAM-1 and VCAM-1 at four hours, and the maximum up-regulatedexpression of ICAM-1 at 16 hours. D2E7 was able to inhibit theexpression of the three adhesion molecules in a dose dependent manner.The IC₅₀ values for the inhibition of ELAM-1, ICAM-1 and VCAM-1 were1.85×10⁻¹⁰, 2.17×10⁻¹⁰ and 1.01×10⁻¹⁰ M, respectively. These values arevery similar, indicating similar requirements for the dose of rhTNFactivation signal to induce ELAM-1, ICAM-1 and VCAM-1 expression.Interestingly, D2E7 was similarly effective in the longer inhibitionassay of the ICAM-1 expression. The ICAM-1 inhibition assay required 16hours of co-incubation of rhTNF and D2E7 with HUVEC as opposed to 4hours required for the ELAM-1 and the VCAM-1 inhibition assays. SinceD2E7 has a slow off-rate for rhTNF, it is conceivable that during the 16hour co-incubation period there was no significant competition by theTNF receptors on the HUVEC.

D. In Vivo Neutralization of hTNFα

Three different in vivo systems were used to demonstrate that D2E7 iseffective at inhibiting hTNFα activity in vivo.

I. Inhibition of TNF-Induced Lethality in D-Galactosamine-SensitizedMice

Injection of recombinant human TNFα (rhTNFα) to D-galactosaminesensitized mice causes lethality within a 24 hour time period. TNFαneutralizing agents have been shown to prevent lethality in this model.To examine the ability of human anti-hTNFα antibodies to neutralizehTNFα in vivo in this model, C57B1/6 mice were injected with varyingconcentrations of D2E7-IgG1, or a control protein, in PBSintraperitoneally (i.p.). Mice were challenged 30 minutes later with 1μg of rhTNFα and 20 mg of D-galactosamine in PBS i.p., and observed 24hours later. These amount of rhTNFα and D-galactosamine were previouslydetermined to achieve 80-90% lethality in these mice.

Representative results, depicted as a bar graph of % survival versusantibody concentration, are shown in FIG. 6. The black bars representD2E7, whereas the hatched bars represent MAK 195. Injection of 2.5-25 μgof D2E7 antibody per mouse protected the animals from TNFα-inducedlethality. The ED₅₀ value is approximately 1-2.5 μg/mouse. The positivecontrol antibody, MAK 195, was similar in its protective ability.Injection of D2E7 in the absence of rhTNFα did not have any detrimentaleffect on the mice. Injection of a non-specific human IgG1 antibody didnot offer any protection from TNFα-induced lethality.

In a second experiment, forty-nine mice were divided into 7 equalgroups. Each group received varying doses of D2E7 thirty minutes priorto receiving an LD₈₀ dose of rhTNF/D-galactosamine mixture (1.0 μg rhTNFand 20 mg D-galactosamine per mouse). Control group 7 received normalhuman IgG1 kappa antibody at 25 μg/mouse dose. The mice were examined 24hours later. Survival for each group is summarized below in Table 13.

TABLE 13 24 Hour Survival After Treatment with D2E7 Group Survival(alive/total) Survival (%) 1 (no antibody) 0/7 0 2 (1 μg) 1/7 14 3 (2.6μg) 5/7 71 4 (5.2 μg) 6/7 86 5 (26 μg) 6/7 86 6 (26 μg; no rhTNF) 7/7100 7 (25 μg Hu IgG1) 1/7 14

II. Inhibition of TNF-Induced Rabbit Pyrexia

The efficacy of D2E7 in inhibiting rhTNF-induced pyrexia response inrabbits was examined. Groups of three NZW female rabbits weighingapproximately 2.5 kg each were injected intravenously with D2E7, rhTNF,and immune complexes of D2E7 and rhTNF. Rectal temperatures weremeasured by thermistor probes on a Kaye thermal recorder every minutefor approximately 4 hours. Recombinant human TNF in saline, injected at5 μg/kg, elicited a rise in temperature greater than 0.4° C. atapproximately 45 minutes after injection. The antibody preparation byitself, in saline at a dose of 138 μg/kg, did not elicit a rise intemperature in the rabbits up to 140 minutes after administration. Inall further experiments, D2E7 or control reagents (human IgG1 or asaline vehicle) were injected i.v. into rabbits followed 15 minuteslater by an injection of rhTNF in saline at 5 μg/kg i.v. Representativeresults of several experiments are summarized below in Table 14:

TABLE 14 Inhibition of rhTNF-induced Pyrexia with D2E7 in Rabbits D2E7Temp. rise*, ° C. Peak Temp. dose rhTNF + Molar Ratio minutes (μg/kg)rhTNF D2E7 % Inhib.** D2E7:rhTNF post rhTNF 14 0.53 0.25 53 1 60 24 0.430.13 70 1.6 40 48 0.53 0.03 94 3.3 50 137 0.53 0.00 100 9.5 60 792 0.800.00 100 55 60 *= Peak temperature **= % inhibition = (1 − {temperaturerise with rhTNF & D2E7/temperature rise with rhTNF alone}) × 100.

Intravenous pretreatment with D2E7 at a dose of 14 μg/kg partiallyinhibited the pyrogenic response, compared to rabbits pre-treated withsaline alone. D2E7 administered at 137 μg/kg totally suppressed thepyrogenic response of rhTNF in the same experiment. In a secondexperiment, D2E7 administered at 24 μg/kg also partially suppressed thepyrogenic response, compared to rabbits pretreated with saline alone.The molar ratio of D2E7 to rhTNF was 1/6:1 in this experiment. In athird experiment, D2E7 injected i.v. at 48 μg/kg (molar ratioD2E7:rhTNF=3.3:1) totally suppressed the pyrogenic response, compared torabbits pretreated with the control human IgG1 in saline at 30 μg/kg. Inthe final experiment, rabbits pretreated with D2E7 (792 μg/kg) at a veryhigh molar ratio to rhTNF (55:1) did not develop any rise in temperatureat any time up to 4 hours of observation. Treatment of rabbits withimmune complexes generated from a mixture of D2E7 and rhTNF incubated at37° C. for 1 hour at a molar ratio of 55:1, without subsequent rhTNFadministration, also did not elicit any rise in temperature in the sameexperiment.

III. Prevention of Polyarthritis in Tg197 Transgenic Mice

The effect of D2E7 on disease development was investigated in atransgenic murine model of arthritis. Transgenic mice (Tg197) have beengenerated that express human wild type TNF (modified in the 3′ regionbeyond the coding sequences) and these mice develop chronicpolyarthritis with 100% incidence at 4-7 weeks of age (see EMBO J (1991)10:4025-4031 for further description of the Tg197 model ofpolyarthritis).

Transgenic animals were identified by PCR at 3 days of age Litters oftransgenic mice were divided into six groups. Transgenic mice wereverified by slot-blot hybridization analysis at 15 days of age. Thetreatment protocols for the six groups were as follows: Group 1=notreatment; Group 2=saline (vehicle); Group 3=D2E7 at 1.5 μg/g; Group4=D2E7 at 15 μg/g; Group 5=D2E7 at 30 μg/g; and Group 6=IgG1 isotypecontrol at 30 μg/g. A litter with non transgenic mice was also includedin the study to serve as a control (Group 7-nontransgenic; notreatment). Each group received three i.p. injections per week of theindicated treatments. Injections continued for 10 weeks. Each week,macroscopic changes in joint morphology were recorded for each animal.At 10 weeks, all mice were sacrificed and mouse tissue was collected informalin. Microscopic examination of the tissue was performed.

Animal weight in grams was taken for each mouse at the start of eachweek. At the same time measurements of joint size (in mm) were alsotaken, as a measurement of disease severity. Joint size was establishedas an average of three measurements on the hind right ankle using amicrometer device. Arthritic scores were recorded weekly as follows:0=No arthritis, (normal appearance and flexion); +=mild arthritis (jointdistortion); ++=moderate arthritis (swelling, joint deformation) and+++=heavy arthritis (ankylosis detected on flexion and severely impairedmovement). Histopathological scoring based on haematoxylin/eosinstaining of joint sections was based as follows; 0=No detectabledisease; 1=proliferation of the synovial membrane; 2=heavy synovialthickening 3=cartilage destruction and bone erosion.

The effect of D2E7 treatment on the mean joint size of the Tg197transgenic arthritic mice is shown in the graph of FIG. 9. Thehistopathological and arthritic scores of the Tg197 transgenic mice, at11 weeks of age, are summarized below in Table 15:

TABLE 15 Effect of D2E7 on Histopathology and Arthritic Score in Tg197Mice Group Treatment Histopathological Score Arthritic Score 1 none 3(7/70 +++ (7/7) 2 saline 3 (8/8) +++ (8/8) 6 IgG1 control 3 (9/9) +++(7/9) 3 D2E7 at 1.5 μg/g 0 (6/8) 0 (8/8) 4 D2E7 at 15 μg/g 0 (7/8) 0(8/8) 5 D2E7 at 30 μg/g 0 (8/8) 0 (8/8)

This experiment demonstrated that the D2E7 antibody has a definitebeneficial effect on transgenic mice expressing the wild-type human TNF(Tg197) with no arthritis evident after the study period.

E. D2E7 Neutralization of TNFαs from Other Species The bindingspecificity of D2E7 was examined by measuring its ability to neutralizetumor necrosis factors from various primate species and from mouse,using an L929 cytotoxicity assay (as described in Example 4, subsectionA, above). The results are summarized in Table 16 below:

TABLE 16 Ability of D2E7 to Neutralize TNF from Different Species in theL929 Assay IC₅₀ for D2E7 TNFα* Source Neutralization (M)** HumanRecombinant 7.8 × 10⁻¹¹ Chimpanzee LPS-stimulated PBMC 5.5 × 10⁻¹¹baboon Recombinant 6.0 × 10⁻¹¹ marmoset LPS-stimulated PBMC 4.0 × 10⁻¹⁰cynomolgus LPS-stimulated PBMC 8.0 × 10⁻¹¹ rhesus LPS-stimulated PBMC3.0 × 10⁻¹¹ canine LPS-stimulated WBC 2.2 × 10⁻¹⁰ porcine Recombinant1.0 × 10⁻⁷  murine Recombinant >1.0 × 10⁻⁷   

The results in Table 16 demonstrate that D2E7 can neutralize theactivity of five primate TNFαs approximately equivalently to human TNFαand, moreover, can neutralize the activity of canine TNFα (aboutten-fold less well than human TNFα) and porcine and mouse TNFα (about˜1000-fold less well than human TNFα). Moreover, the binding of D2E7 tosolution phase rhTNFα was not inhibited by other cytokines, such aslymphotoxin (TNFβ), IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IFNγ and TGFβ,indicating that D2E7 is very specific for its ligand TNFα.

F. Lack of Cytokine Release by Human Whole Blood Incubated with D2E7

In this example, the ability of D2E7 to induce, by itself, normal humanblood cells to secrete cytokines or shed cell surface molecules wasexamined. D2E7 was incubated with diluted whole blood from threedifferent normal donors at varying concentrations for 24 hours. An LPSpositive control was run at the same time, at a concentration previouslydetermined to stimulate immunocompetent blood cells to secretecytokines. The supernatants were harvested and tested in a panel of tensoluble cytokine, receptor and adhesion molecule ELISA kits: IL-1α,IL-1β, IL-1 receptor antagonist, IL-6, IL-8, TNFα, soluble TNF receptorI, soluble TNF receptor II, soluble ICAM-1 and soluble E-selectin. Nosignificant amounts of cytokines or shed cell surface molecules weremeasured as a result of D2E7 antibody co-incubation, at concentrationsup to 343 μg/ml. Control cultures without the addition of the antibodyalso did not yield any measurable amounts of cytokines, whereas the LPSco-culture control yielded elevated values in the high picogram to lownanogram range. These results indicate that D2E7 did not induce wholeblood cells to secrete cytokines or shed cell surface proteins abovenormal levels in ex vivo cultures.

Forming part of the present disclosure is the appended Sequence Listing,the contents of which are summarized in the table below:

ANTIBODY SEQ ID NO: CHAIN REGION SEQUENCE TYPE 1 D2E7 VL amino acid 2D2E7 VH amino acid 3 D2E7 VL CDR3 amino acid 4 D2E7 VH CDR3 amino acid 5D2E7 VL CDR2 amino acid 6 D2E7 VH CDR2 amino acid 7 D2E7 VL CDR1 aminoacid 8 D2E7 VH CDR1 amino acid 9 2SD4 VL amino acid 10 2SD4 VH aminoacid 11 2SD4 VL CDR3 amino acid 12 EP B12 VL CDR3 amino acid 13 VL10E4VL CDR3 amino acid 14 VL100A9 VL CDR3 amino acid 15 VLL100D2 VL CDR3amino acid 16 VLL0F4 VL CDR3 amino acid 17 LOE5 VL CDR3 amino acid 18VLLOG7 VL CDR3 amino acid 19 VLLOG9 VL CDR3 amino acid 20 VLLOH1 VL CDR3amino acid 21 VLLOH10 VL CDR3 amino acid 22 VL1B7 VL CDR3 amino acid 23VL1C1 VL CDR3 amino acid 24 VL0.1F4 VL CDR3 amino acid 25 VL0.1H8 VLCDR3 amino acid 26 LOE7.A VL CDR3 amino acid 27 2SD4 VH CDR3 amino acid28 VH1B11 VH CDR3 amino acid 29 VH1D8 VH CDR3 amino acid 30 VH1A11 VHCDR3 amino acid 31 VH1B12 VH CDR3 amino acid 32 VH1E4 VH CDR3 amino acid33 VH1F6 VH CDR3 amino acid 34 3C-H2 VH CDR3 amino acid 35 VH1-D2.N VHCDR3 amino acid 36 D2E7 VL nucleic acid 37 D2E7 VH nucleic acid

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. Use of an isolated human antibody, or an antigen-binding portionthereof, that dissociates from human TNFα with a K_(d) of 1×10⁻⁸ M orless and a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determinedby surface plasmon resonance, and neutralizes human TNFα cytotoxicity ina standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁷ M or less, incombination with at least one additional therapeutic agent for treatinga disorder in which TNFα activity is detrimental.
 2. The use of claim 1,wherein the isolated human antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) rate constant of 5×10⁻⁴ s⁻¹or less.
 3. The use of claim 1, wherein the isolated human antibody, orantigen-binding portion thereof, dissociates from human TNFα with aK_(off) rate constant of 1×10⁻⁴ s⁻¹ or less.
 4. The use of claim 1,wherein the isolated human antibody, or antigen-binding portion thereof,neutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻⁸ M or less.
 5. The use of claim 1, wherein theisolated human antibody, or antigen-binding portion thereof, neutralizeshuman TNFα cytotoxicity in a standard in vitro L929 assay with an IC₅₀of 1×10⁻⁹ M or less.
 6. The use of claim 1, wherein the isolated humanantibody, or antigen-binding portion thereof, neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 5×10⁻¹⁰ Mor less.
 7. The use of claim 1, wherein the isolated human antibody, orantigen-binding portion thereof, is a recombinant antibody, orantigen-binding portion thereof.
 8. The use of claim 1, wherein theadditional therapeutic agent is selected from the group consisting ofnon-steroidal anti-inflammatory drugs, cytokine suppressiveanti-inflammatory drugs, CDP-571/BAY-10-3356, cA2, 75 kdTNFR-IgG, 55kdTNFR-IgG, IDEC-CE9.1/SB 210396, DAB 486-IL-2, DAB 389-IL-2, Anti-Tac,IL-4, IL-10, IL-4 agonists, IL-10 agonists, IL-1RA, TNF-bp/s-TNFR, S284,R973401, MK-966, Iloprost, methotrexate, thalidomide,thalidomide-related drugs, leflunomide, tranexamic acid, T-614,prostaglandin E1, Tenidap, Naproxen, Meloxicam, Piroxicam, Diclofenac,Indomethacin, Sulfasalazine, Azathioprine, ICE inhibitors, zap-70inhibitors, lck inhibitors, VEGF inhibitors, VEGF-R inhibitors,corticosteroids, TNF-convertase inhibitors, anti-L-12 antibodies,interleukin-11, interleukin-13, interleukin-17 inhibitors, gold,penicillamine, chloroquine, hydroxychloroquine, chlorambucil,cyclophosphamide, cyclosporin, anti-thymocyte globulin, anti-CD4antibodies, CD5-toxins, orally-administered peptides, collagen,lobenzarit disodium, Cytokine Regulating Agents HP228 and HP466, ICAM-1antisense phosphorothioate oligodeoxynucleotides, soluble complementreceptor 1, prednisone, orgotein, glycosaminoglycan polysulphate,minocycline, anti-IL2R antibodies, marine lipids, botanical lipids,auranofin, phenylbutazone, meclofenamic acid, flufenamic acid,intravenous immune globulin, zileuton, mycophenolic acid, tacrolimus,sirolimus, amiprilose, cladribine, azaribine, budenoside, epidermalgrowth factor, aminosalicylates, 6-mercaptopurine, metronidazole,lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide,antioxidants, thromboxane inhibitors, IL-1 receptor antagonists,anti-IL-1β monoclonal antibodies, anti-IL-6 monoclonal antibodies,growth factors, elastase inhibitors, pyridinyl-imidazole compounds,glucuronide-conjugated prodrugs of prednisolone, dexamethasone orbudesonide, dextran-conjugated prodrugs of prednisolone, dexamethasoneor budesonide, soluble complement receptor 1, slow-release mesalazine,antagonists of Platelet Activating Factor (PAF), ciprofloxacin,lignocaine, prednisolone, methylprednisolone, cyclophosphamide,4-aminopyridine, tizanidine, interferon-β1a, interferon-β1b, Copolymer1, hyperbaric oxygen, intravenous immunoglobulin, clabribine, hypertonicsaline solutions, antibiotics, continuous hemofiltration, carbapenems,antagonists of cytokines such as TNFα, L-1β, IL-6 and/or IL-8, SK&F107647, tetravalent guanylhydrazone CNI-1493, Tissue Factor PathwayInhibitor, PHP, iron chelators and chelates, includingdiethylenetriamine pentaacetic acid—iron (III) complex, lisofylline,PGG-Glucan, apolipoprotein A-1 reconstituted with lipids, chiralhydroxamic acids, anti-endotoxin antibodies, E5531, rBPI₂₁, SyntheticAnti-Endotoxin Peptides, surfactant replacement therapy and anti-IL-8antibodies.
 9. The use of claim 1, wherein the disorder in which TNFαactivity is detrimental is rheumatoid arthritis.